Zwitterionically modified polymers and hydrogels

ABSTRACT

The present invention is directed to a polymer of Formula (IV):wherein A, X, Q, Y, Z, m1, m2, m3, k1, and k2 are as described herein and wherein the monomer units of the polymer are the same or different. The present invention also relates to a monomer of Formula (III):wherein R″, X1, Y1, Z1, m4, m5, and m6 are as described herein, and a polymeric network comprising two or more monomers of Formula (III). The present invention also relates to a hydrogel comprising any of the polymers and monomers described herein, a capsule comprising the hydrogel, and a method of delivering a therapeutic agent to a subject using the capsule.

This application is a continuation of U.S. patent application Ser. No.16/480,996 filed Jul. 25, 2019, which is a national stage applicationunder 35 U.S.C. § 371 of International Application No.PCT/US2018/015613, filed Jan. 27, 2018, which claims the benefit of U.S.Provisional Patent Application Ser. No. 62/451,629, filed Jan. 27, 2017,which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a zwitterionic monomers, polymers, andhydrogels, and their use to coat and/or encapsulate biologicalmaterials.

BACKGROUND OF THE INVENTION

Although many advanced therapeutic cell treatments have been developedrecently, type 1 diabetes (T1D) is still a global epidemic affectingover millions of people worldwide. Pancreatic islet transplantation hasbeen considered as an alternative and promising approach for thetreatment of T1D. However, islet transportation in the clinic is limitedby two major hurdles: shortage of donor islets and long-termimmuno-suppression. Recently, human stem cell-derived beta cells havebeen developed, providing a pathway to produce an unlimited supply ofinsulin-producing cells. Therefore, there is a critical need fordevelopment of novel materials or medical devices that encapsulateislets to protect them from the host immune response effectively.

The performance of implanted biomaterials is often impeded by theforeign body response (FBR), which leads to the formation of a densecollagenous capsule and then the failure of the medical device.Nonspecific protein adsorption on the implanted material is consideredthe first step of the foreign body response. Incorporation of anantifouling material or a surface that highly resists protein adsorptionand cell attachment is expected to suppress FBR and the subsequentformation of a fibrotic capsule. Recently, the use of zwitterionicpolymers, bearing zwitterion of carboxybetaine, sulfobetaine, andphosphorycholine, have drawn much attention due to the ultra-low-foulingproperties of these polymers (Jiang et al., “Ultralow-Fouling,Functionalizable, and Hydrolyzable Zwitterionic Materials and TheirDerivatives for Biological Applications,” Advanced Materials 22(9):920-932 (2010)). For example, zwitterionic poly(carboxybetainemethacrylate) (PCBMA) has shown to resist the formation of a capsule forat least 3 months after subcutaneous implantation in mice (Zhang et al.,“Zwitterionic Hydrogels Implanted in Mice Resist the Foreign-BodyReaction,” Nature Biotechnology 31(6):553-556 (2013)). However, theharsh conditions associated with gelation of the zwitterionic materials,e.g., UV irradiation and free radical generation, can cause much harm tothe encapsulated cell, limiting the applications of these materials forcell encapsulation.

The naturally derived material, alginate, has also shown potential foruse in numerous applications including tissue regeneration, drugdelivery and cell encapsulation, due in a large part to its mildgelation and low toxicity (Drury et al., “The Tensile Properties ofAlginate Hydrogels,” Biomaterials 25(16):3187-3199 (2004)). However,alginate elicits a fibrotic response which is worsened with theencapsulation of cells or xenogeneic donor tissue. The fibrotic tissueon the alginate surface cuts off the diffusion of nutrients and oxygento the encapsulated cell, causing cell necrosis. Thus there is a need inthe art for a polymeric material that does not elicit the FBR andpromotes the health of the encapsulated cells or tissue.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

A first aspect of the present invention is directed to a monomer ofFormula (I):

wherein

-   -   A is selected from a saccharide containing unit and a polyvinyl        alcohol containing unit;    -   X is selected from the group consisting of O, NH, NR′, C(O), and        C₁₋₂₀ alkylene, wherein C₁₋₂₀ alkylene is optionally substituted        from 1 to 20 times with a substituent selected independently at        each occurrence thereof from the group consisting of OH,        halogen, cyano, —CF₃, and C₁₋₆ alkoxy.    -   Q is absent or is a linker;    -   Y is selected from the group consisting of

-   -   Z is selected from the group consisting of

-   -   m₁ is 0 to 50;    -   m₂ is 0 to 50;    -   m₃ is 0 to 50;    -   R is C₁₋₂₀ alkyl;    -   R′ is —C(O)—C₁₋₆ alkene;    -   R¹ is C₁₋₂₀ alkyl;    -   R² is C₁₋₂₀ alkyl; and    -   R³ is C₁₋₂₀ alkyl.

Another aspect of the present invention relates to a polymer of Formula(IV):

wherein

-   -   A is independently selected from a saccharide containing unity        and a polyvinyl alcohol containing unit for each monomer unit of        the polymer;    -   X is selected from the group consisting of O, NH, NR′, C(O), and        C₁₋₂₀ alkylene, wherein C₁₋₂₀ alkylene is optionally substituted        from 1 to 20 times with a substituent selected independently at        each occurrence thereof from the group consisting of OH,        halogen, cyano, —CF₃, and C₁₋₆ alkoxy    -   Q is absent or is a linker;    -   Y is selected from the group consisting of

-   -   Z is selected from the group consisting of

-   -   m₁ is 0 to 50;    -   m₂ is 0 to 50;    -   m₃ is 0 to 50;    -   R is C₁₋₂₀ alkyl;    -   R′ is —C(O)—C₁₋₆ alkene;    -   R¹ is C₁₋₂₀ alkyl;    -   R² is C₁₋₂₀ alkyl;    -   R³ is C₁₋₂₀ alkyl,    -   k₁ is any integer; and    -   k₂ is independently selected for each monomer unit from 0 or 1,        with the proviso that at least one k₂ is 1;    -   wherein the monomer units of the polymer are the same or        different.

Another aspect of the present invention relates to a polymer, whereinsaid polymer comprises one or more monomers of Formula (I):

and further comprises

-   -   one or more monomers of Formula (II):

A-L¹-L²-L³-R⁴  (II)

-   -   wherein    -   L¹ is selected from the group consisting of O, NH, NR′, C(O),        and C₁₋₂₀ alkylene, wherein C₁₋₂₀ alkylene is optionally        substituted from 1 to 20 times with a substituent selected        independently at each occurrence thereof from the group        consisting of OH, halogen, cyano, —CF₃, and C₁₋₆ alkoxy;    -   L² is absent or is C₁₋₂₀ alkylene, wherein C₁₋₂₀ alkylene is        optionally substituted from 1 to 20 times with a substituent        selected independently at each occurrence thereof from the group        consisting of OH, halogen, cyano, —CF₃, and C₁₋₆ alkoxy;    -   L³ is selected from the group consisting of C₁₋₂₀ alkylene,        C₁₋₂₀ alkenylene, C₃₋₁₂ cycloalkenylene, and arylene, wherein        arylene is optionally substituted from 1 to 3 times with a        substituent selected independently at each occurrence thereof        from the group consisting of heteroarylene and heterocyclylene;        and    -   R⁴ is selected from the group consisting of H, SH, N₃, C₁₋₆        alkyl, C₂₋₂₀ alkenyl, C₂₋₂₀ alkynyl, C₃₋₁₂ cycloalkenyl, C₃₋₁₂        cycloalkynyl, heteroaryl, and heterocyclyl, wherein C₁₋₆ alkyl,        C₂₋₂₀ alkenyl, C₂₋₂₀ alkynyl, C₃₋₁₂ cycloalkenyl, C₃₋₁₂        cycloalkynyl, heteroaryl, and heterocyclyl is optionally        substituted from 1 to 3 times with a substituent selected        independently at each occurrence thereof from the group        consisting of H, OH, halogen, cyano, —CF₃, and C₁₋₆ alkoxy.

Another aspect of the present invention relates to a monomer of Formula(III):

-   -   wherein    -   X is absent or is

-   -   Y¹ is

-   -   Z¹ is

-   -   m₄ is 1 to 50;    -   m₅ is 0 to 10;    -   m₆ is 1 to 50;    -   R″ is H or C₁₋₆ alkyl;    -   R⁴ is C₁₋₂₀ alkyl;    -   R⁵ is C₁₋₂₀ alkyl; and    -   R⁶ is C₁₋₂₀ alkyl,    -   with the proviso that when X¹ is absent, Y¹ is not

Yet another aspect of the present invention relates to a polymericnetwork comprising crosslinked monomers of Formula (III):

Another aspect of the present invention relates to a hydrogel comprisingany one of the polymers as described herein, the polymeric network asdescribed herein, or any combination thereof.

Another aspect of the present invention relates to a capsule comprisinga hydrogel of the present invention and a therapeutic agent encapsulatedin the hydrogel.

Another aspect of the present invention is directed to a method ofdelivering a therapeutic agent to a subject. This method involvesadministering, to a subject, a capsule comprising the hydrogel and atherapeutic agent encapsulated by the hydrogel as described herein.

A further aspect of the present invention is directed to a method oftreating a diabetic subject. This method involves implanting, into asubject having diabetes, a capsule comprising a therapeutic agent asdescribed herein.

Alginates and other polymers chemically modified with ultra-low fouling,zwitterionic groups such as sulfobetaine and carboxybetaine as describedherein exhibit superior biocompatibility. The zwitterionic moietyendowed alginate with excellent long-term biocompatibility andsuppressed the fibrotic response, while the alginate backbone remainscrosslinkable under mild gelation condition. These materials areparticularly suitable for cell encapsulation and transplantation as wellas in other biological applications using alginate and hydrogels. Thisis the first work to develop zwitterion-based alginate conjugates, whichhave applicability in areas such as islet encapsulation for type 1diabetes treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show ¹H NMR of spectrums of the sulfobetaine-NH₂ (FIG. 1A)and carboxybetaine-NH₂ (FIG. 1B) monomers.

FIGS. 2A-2B show synthesis and characterization of thesulfobetaine-based alginate conjugates as described herein. FIG. 2Ashows synthetic pathway of the sulfobetaine-based alginate conjugates.FIG. 2B shows ¹H NMR spectrum of alginate, sulfobetaine-based alginateconjugate, and sulfobetaine-NH₂ monomer in D₂O.

FIGS. 3A-3B show synthesis and characterization of thecarboxybetaine-based alginate conjugates as described herein. FIG. 3Ashows synthetic pathway of the carboxybetaine-based alginate conjugates.FIG. 3B shows ¹H NMR spectrum of alginate, carboxybetaine-based alginateconjugate, and carboxybetaine-NH₂ monomer in D₂O.

FIG. 4 are images showing three kinds of sulfobetaine-modified alginatemicrocapsules (SB-VLG, SB-SLG20, and SB-SLG100) with almost no fibrosisafter 14 days of intraperitoneal implantation in C57BL/6J mice.Dark-field phase contrast images of the microcapsules retrieved after 2weeks in the intraperitoneal space show much less fibrosis onSB-alginate microcapsules than on SLG20 unmodified control microcapsule.SLG20, VLVG, SLG100 are alginates with different molecular weights.Scale bars, 2000 μm; n=5; each image represents one mouse.

FIG. 5 shows sulfobetaine (SB) modified alginate (SB-SLG 20)microcapsules with almost no fibrosis after 100 days of intraperitonealimplantation in C57BL/6J mice. Dark-field phase contrast images of themicrocapsules retrieved after 100 days in the intraperitoneal space showmuch less fibrosis on SB modified microcapsules (bottom row of images)than on SLG20 control microcapsules (top row of images). Scale bars,2000 μm; each image represents one mouse, n=4.

FIG. 6 shows SB-SLG 20 microcapsules with almost no fibrosis after 180days of intraperitoneal implantation in C57BL/6J mice. Dark-field phasecontrast images of the microcapsules retrieved after 180 days in theintraperitoneal space show much less fibrosis on SB modifiedmicrocapsules than on SLG20 control microcapsule. Scale bars, 2000 μm;each image represents one mouse, n=7.

FIG. 7 shows images of the retrieved SB-SLG 20 (bottom two rows) and SLG20 (top two rows) microcapsules after 180 days of intraperitonealimplantation in C57BL/J6 mice. n=7. The whiteness of the capsulesindicates fibrosis.

FIG. 8 shows dark-field phase contrast images of ethylene-glycol SBmicrocapsules with almost no fibrosis after 30 days of intraperitonealimplantation in C57BL/6J mice. Scale bars, 2000 μm; n=5.

FIGS. 9A-9G show that rat islets encapsulated in SB-SLG 20 capsulesmaintained long-term normoglycemia in STZ-treated diabetic C57BL/6J miceas compared to the SLG20 control group. FIG. 9A is a representativedark-field phase contrast image of encapsulated rat islets in 1000 μmSB-SLG20 microcapsules. Scale bars, 500 μm. FIG. 9B is a graph showingblood glucose concentrations of mice from 3-month transplantationstudies (n=3 mice per treatment group). FIG. 9C is representativedark-field phase contrast image (scale bars, 2000 μm) and FIG. 9D is anH&E stained cross-sectional image of retrieved islet-containing SLG 20microcapsules after 90 days implantation. The whiteness of the capsuleindicates the presence of fibrosis. FIG. 9E is a representativedark-field phase contrast image (scale bars, 2000 μm) and FIG. 9F is anH&E stained cross-sectional image of retrieved islet-containing SB-SLG20 microcapsules after 90 days implantation. Note the absence offibrosis on the capsules and numerous islets inside the capsules. FIG.9G is an image showing immunohistochemical staining of islets (DAPI) inretrieved SB-SLG 20 microcapsules. Insulin also visualized with stain;scale bars, 50 μm.

FIG. 10 shows ¹H NMR spectrum of product 10a at 400 MHz, CDCl₃.

FIG. 11 shows ¹H NMR spectrum of product 15a at 400 MHz, CDCl₃.

FIG. 12 shows ¹H NMR spectrum of product 16a at 400 MHz, CDCl₃.

FIG. 13 shows ¹H NMR spectrum of product 17a at 400 MHz, D₂O.

FIG. 14 shows ¹H NMR spectrum of qTR-CB at 400 MHz, D₂O.

FIG. 15 shows ¹H NMR spectrum of product 9a at 400 MHz, D₂O.

FIG. 16 shows ¹H NMR spectrum of product 11a at 400 MHz, D₂O.

FIG. 17 shows ¹H NMR spectrum of TR-CB at 400 MHz, D₂O.

FIG. 18 shows ¹H NMR spectrum of product 12a at 400 MHz, CDCl₃.

FIG. 19 shows ¹H NMR spectrum of TR-SB at 400 MHz, D₂O.

FIGS. 20A-20D show synthesis and characterization of the qTR-CB. FIG.20A shows the chemical structure of qTR-CB. FIG. 20B shows syntheticroute of qTR-CB. FIG. 20C shows typical surface plasmon resonance (SPR)sensorgrams showing protein adsorption from 1 mg/mL fibrinogen (Fg) orundiluted human plasma on the P(qTR-CB)-grafted or bare gold surfaces.Lines from top to bottom at 15 minutes are in the same order as thelegend. FIG. 20D is an image of a P(qTR-CB) hydrogel.

FIGS. 21A-21E show mechanical properties of the P(qTR-CB) hydrogel. FIG.21A is a schematic illustration showing the π-π stacking between thetriazole rings as potential mechanism for energy dissipation. FIG. 21Bshows images of poly(carboxybetaine) (PCB) and P(qTR-CB) hydrogelsduring folding test. FIG. 21C is a graph showing stress-strain curvesfor PCB and P(qTR-CB) hydrogels in tensile test. FIG. 21D is a graphshowing stress-strain curves for PCB and P(qTR-CB) hydrogels incompression test. FIG. 21E is a graph showing stress-strain curves often consecutive loading-unloading cycles for the P(qTR-CB) hydrogel.

FIGS. 22A-22B show in vitro characterization of the P(qTR-CB) hydrogel.FIG. 22A shows fluorescent microscopic images of NIH/3T3 cells after3-days of culturing on tissue culture polystyrene (TCPS),poly(2-hydroxyethyl methacrylate) (PHEMA), PCB, and P(qTR-CB) hydrogelsurfaces (Scale bars: 100 μm), and quantification of the cell density(Mean±s.d; n=5; *, p<0.05; ns, not significant). FIG. 22B is a graphshowing quantification of TNF-α and IL10 secretion from macrophagescultured on various surfaces. (Mean±s.d; n=6; *, p<0.05; ns, notsignificant). The order of bars in each condition of each graph fromleft to right: TCPS, PHEMA, PCB, P(qTR-CB).

FIGS. 23A-23E show synthesis of P(TR-CB) and P(TR-SB) hydrogels andcharacterizations of their mechanical properties. FIG. 23A showssynthetic routes to TR-CB and TR-SB monomers. FIG. 23B shows images ofP(TR-CB) and P(TR-SB) hydrogels during folding test. FIG. 23C showsstretching of a P(TR-SB) hydrogel. FIG. 23D is a graph showingstress-strain curves for the PCB, P(TR-CB), and P(TR-SB) hydrogels intensile test. FIG. 23E is a graph showing stress-strain curves for thePCB, P(TR-CB), and P(TR-SB) hydrogels in compression test.

FIGS. 24A-24B show characterization of the foreign body response (FBR)to various hydrogels in immunocompetent mice. FIG. 24A showsrepresentative Masson's trichrome staining images of different hydrogelsretrieved at the indicated time points after subcutaneous implantation.The blue staining indicates fibrosis or collagen deposition surroundingimplants (Scale bars: 100 μm; asterisks indicate the location of theimplanted hydrogel). FIG. 24B are graphs showing quantification ofcollagen density around the implants (n=5).

FIG. 25 shows fluorescence microscopic images of NIH/3T3 cells attachedafter 3-days culturing on P(TR-CB) and P(TR-SB) hydrogel surfaces (Scalebars: 100 μm).

FIG. 26A-26B show characterization of FBR to various hydrogels inimmunocompetent mice after 1 month subcutaneous implantation. FIG. 26A(left) shows representative Masson's trichrome stain image of PCBhydrogel (the blue staining indicates fibrosis or collagen depositionsurrounding implants; scale bar: 100 μm; asterisks indicate the locationof the implanted hydrogel). The graph of FIG. 26A (right) showsquantification of collagen density around the implants. FIG. 26B (left)shows representative CD31 immunostaining image of PCB hydrogel (bloodvessels are stained dark green and nuclei are stained blue; scale bars:50 μm; asterisks indicate the location of the implanted hydrogel anddashed lines indicate the border between the fibrotic layer and the skintissue). The graph of FIG. 26B (right) shows quantification of bloodvessel density around the implants. All data are presented as meanvalue±s.d. (Five mice per type of hydrogel); *, P<0.05; ns, notsignificant.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to a monomer of Formula (I):

wherein

-   -   A is selected from a saccharide containing unit and a polyvinyl        alcohol containing unit;    -   X is selected from the group consisting of O, NH, NR′, C(O), and        C₁₋₂₀ alkylene, wherein C₁₋₂₀ alkylene is optionally substituted        from 1 to 20 times with a substituent selected independently at        each occurrence thereof from the group consisting of OH,        halogen, cyano, —CF₃, and C₁₋₆ alkoxy    -   Q is optional, and if present is a linker;    -   Y is selected from the group consisting of

-   -   Z is selected from the group consisting of

-   -   m₁ is 0 to 50;    -   m₂ is 0 to 50;    -   m₃ is 0 to 50;    -   R is C₁₋₂₀ alkyl;    -   R′ is —C(O)—C₁₋₆ alkene;    -   R¹ is C₁₋₂₀ alkyl;    -   R² is C₁₋₂₀ alkyl; and    -   R³ is C₁₋₂₀ alkyl.

In one embodiment, Q is absent from the monomer of the presentinvention. In another embodiment, Q is present as a linker in themonomer of the present invention. Q is an organic grouping containingany number of carbon atoms, 1-30 carbon atoms, 1-20 carbon atoms, or1-14 carbon atoms, and optionally including one or more heteroatoms suchas nitrogen, sulfur, or nitrogen in linear, branched, or cyclicstructural formats. Representative Q groupings being C₁₋₂₀ alkylene,substituted C₁₋₂₀ alkylene, phenoxy, substituted phenoxy, alkoxy,substituted alkoxy, C₃₋₂₀ cycloalkylene, substituted C₃₋₂₀cycloalkylene, C₃₋₂₀ cycloalkenylene, substituted C₃₋₂₀ cycloalkenylene,C₈₋₂₀ cycloalkynylene, substituted C₈₋₂₀ cycloalkynylene,heterocyclylene, substituted heterocyclylene, arylene, substitutedarylene, triazole, poly(ethylene glycol), or polypeptide moiety.

In one embodiment, Q is selected from the group consisting of C₁₋₂₀alkylene, C₃₋₂₀ cycloalkylene, arylene, heteroarylene, heterocyclene,—O—C₁₋₂₀ alkylene, poly(ethylene glycol), and polypeptide, wherein C₁₋₂₀alkylene, C₃₋₂₀ cycloalkylene, arylene, heteroarylene, heterocyclylene,or —O—C₁₋₂₀ alkylene is optionally substituted from 1 to 20 times with asubstituent selected independently at each occurrence thereof from thegroup consisting of —OH, halogen, cyano, —CF₃, C₁₋₆ alkyl, and C₁₋₆alkoxy, and wherein C₁₋₂₀ alkylene is optionally interrupted by one ormore heteroatoms selected from the group consisting of oxygen nitrogen,sulfur, or nitrogen.

In another embodiment, Q is heteroarylene.

In yet another embodiment, Q is

As used above, and throughout the description herein, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings. If not defined otherwise herein, all technical andscientific terms used herein have the same meaning as is commonlyunderstood by one of ordinary skill in the art to which this technologybelongs. In the event that there is a plurality of definitions for aterm herein, those in this section prevail unless stated otherwise.

The term “alkyl” means an aliphatic hydrocarbon group which may bestraight or branched having about 1 to about 20 (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) carbon atoms in thechain, unless otherwise specified. Branched means that one or more loweralkyl groups such as methyl, ethyl, or propyl are attached to a linearalkyl chain. Exemplary alkyl groups include methyl, ethyl, n-propyl,i-propyl, n-butyl, t-butyl, n-pentyl, and 3-pentyl.

The term “alkylene” means a group obtained by removal of a hydrogen atomfrom an alkyl group. An alkylene is a divalent, straight or branchedchain alkane group. Non-limiting examples of alkylene include methyleneand ethylene.

The term “alkenyl” means an aliphatic hydrocarbon group containing acarbon-carbon double bond and which may be straight or branched havingabout 2 to about 20 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20) carbon atoms in the chain. Preferred alkenylgroups have 2 to about 6 (e.g., 2, 3, 4, 5, 6) carbon atoms in thechain. Exemplary alkenyl groups include ethenyl, propenyl, n-butenyl,and i-butenyl. An alkenylene is a divalent, straight or branched chainalkene group.

The term “alkynyl” means an aliphatic hydrocarbon group containing acarbon-carbon triple bond and which may be straight or branched havingabout 2 to about 20 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20) carbon atoms in the chain. Preferred alkynylgroups have 2 to about 6 (e.g., 2, 3, 4, 5, 6) carbon atoms in thechain. Exemplary alkynyl groups include ethynyl, propynyl, n-butynyl,2-butynyl, 3-methylbutynyl, and n-pentynyl. An alkynylene is a divalent,straight or branched chain alkyne.

The term “cycloalkyl” refers to a non-aromatic saturated mono- orpolycyclic ring system which may contain 3 to 20 (e.g., 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) carbon atoms.Exemplary cycloalkyl groups include, without limitation, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl.

The term “cycloalkylene” means a group obtained by removal of a hydrogenatom from a cycloalkyl group. Non-limiting examples of cycloalkyleneinclude cyclobutylene and cyclopropylene.

The term “cycloalkenyl” refers to a non-aromatic unsaturated mono- orpolycyclic ring system which may contain 3 to 12 (e.g., 3, 4, 5, 6, 7,8, 9, 10, 11, or 12) carbon atoms, and which includes at least onedouble bond. Exemplary cycloalkenyl groups include, without limitation,cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl. Acycloalkenylene is a divalent, straight or branched chain cycloalkenegroup.

The term “cycloalkynyl” refers to a non-aromatic unsaturated mono- orpolycyclic ring system which may contain 8 to 12 carbon atoms, and whichincludes at least one triple bond. Exemplary cycloalkynyl groupsinclude, without limitation, cyclononyne and cyclooctyne. Acycloalkynylene is a divalent, straight or branched chain cycloalkynegroup.

As used herein, the term “heterocyclyl” refers to a stable 3- to18-membered (e.g., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-,15-, 16-, 17-, or 18-membered) ring system that consists of carbon atomsand from one to five (e.g., 1, 2, 3, 4, or 5) heteroatoms selected fromthe group consisting of nitrogen, oxygen, sulfur, and silicon. Theheterocyclyl may be a monocyclic or a polycyclic ring system, which mayinclude fused, bridged, or spiro ring systems; and the nitrogen, carbon,sulfur, or silicon atoms in the heterocyclyl may be optionally oxidized;the nitrogen atom may be optionally quaternized; and the ring may bepartially or fully saturated. Representative monocyclic heterocyclylsinclude piperidine, piperazine, pyrimidine, morpholine, thiomorpholine,pyrrolidine, tetrahydrofuran, pyran, tetrahydropyran, oxetane, and thelike. Representative polycyclic heterocyclyls include indole, isoindole,indolizine, quinoline, isoquinoline, purine, carbazole, dibenzofuran,chromene, xanthene, and the like.

The term “heterocyclylene” means a group obtained by removal of ahydrogen atom from a heterocyclyl group. Non-limiting examples ofheterocyclylene include piperidylene, pyrrolidinylene, piperazinylene,morpholinylene, thiomorpholinylene, thiazolidinylene, 1,4-dioxanylene,tetrahydrofuranylene and tetrahydrothiophenylene.

As used herein, the term “aryl” refers to an aromatic monocyclic orpolycyclic ring system containing from 6 to 19 (e.g., 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, or 19) carbon atoms, where the ringsystem may be optionally substituted. Aryl groups of the presentinvention include, but are not limited to, groups such as phenyl,naphthyl, azulenyl, phenanthrenyl, anthracenyl, fluorenyl, pyrenyl,triphenylenyl, chrysenyl, and naphthacenyl.

The term “arylene” means a group obtained by removal of a hydrogen atomfrom an aryl group. Non-limiting examples of arylene include phenyleneand naphthylene.

As used herein, “heteroaryl” refers to an aromatic ring radical whichconsists of carbon atoms and from one to five (e.g., 1, 2, 3, 4, or 5)heteroatoms selected from the group consisting of nitrogen, oxygen,sulfur, and silicon. Examples of heteroaryl groups include, withoutlimitation, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, furyl,thiophenyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl,thiadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl,thienopyrrolyl, furopyrrolyl, indolyl, azaindolyl, isoindolyl,indolinyl, indolizinyl, indazolyl, benzimidazolyl, imidazopyridinyl,benzotriazolyl, benzoxazolyl, benzoxadiazolyl, benzothiazolyl,pyrazolopyridinyl, triazolopyridinyl, thienopyridinyl,benzothiadiazolyl, benzofuyl, benzothiophenyl, quinolinyl,isoquinolinyl, tetrahydroquinolyl, tetrahydroisoquinolyl, cinnolinyl,quinazolinyl, quinolizilinyl, phthalazinyl, benzotriazinyl, chromenyl,naphthyridinyl, acrydinyl, phenanzinyl, phenothiazinyl, phenoxazinyl,pteridinyl, and purinyl. Additional heteroaryls are described inCOMPREHENSIVE HETEROCYCLIC CHEMISTRY: THE STRUCTURE, REACTIONS,SYNTHESIS AND USE OF HETEROCYCLIC COMPOUNDS (Katritzky et al. eds.,1984), which is hereby incorporated by reference in its entirety.

The term “heteroarylene” means a group obtained by removal of a hydrogenatom from a heteroaryl group. Non-limiting examples of heteroaryleneinclude pyridylene, pyrazinylene, furanylene, thienylene andpyrimidinylene.

The term “optionally substituted” is used to indicate that a group mayhave a substituent at each substitutable atom of the group (includingmore than one substituent on a single atom), provided that thedesignated atom's normal valency is not exceeded and the identity ofeach substituent is independent of the others. Up to three H atoms ineach residue are replaced with alkyl, halogen, haloalkyl, hydroxy,loweralkoxy, carboxy, carboalkoxy (also referred to as alkoxycarbonyl),carboxamido (also referred to as alkylaminocarbonyl), cyano, carbonyl,nitro, amino, alkylamino, dialkylamino, mercapto, alkylthio, sulfoxide,sulfone, acylamino, amidino, phenyl, benzyl, heteroaryl, phenoxy,benzyloxy, or heteroaryloxy. “Unsubstituted” atoms bear all of thehydrogen atoms dictated by their valency. When a substituent is keto(i.e., ═O), then two hydrogens on the atom are replaced. Combinations ofsubstituents and/or variables are permissible only if such combinationsresult in stable compounds; by “stable compound” or “stable structure”is meant a compound that is sufficiently robust to survive isolation toa useful degree of purity from a reaction mixture, and formulation intoan efficacious therapeutic agent.

The term “substituted” or “substitution” means that one or more hydrogenon a designated atom is replaced with a selection from the indicatedgroup, provided that the designated atom's normal valency is notexceeded. “Unsubstituted” atoms bear all of the hydrogen atoms dictatedby their valency. When a substituent is oxo (i.e., ═O), then 2 hydrogenson the atom are replaced. Combinations of substituents and/or variablesare permissible only if such combinations result in stable compounds. By“stable compound” it is meant a compound that is sufficiently robust tosurvive isolation to a useful degree of purity from a reaction mixture,and formulation into an efficacious therapeutic agent. Exemplarysubstituents include, without limitation, oxo, thio (i.e., ═S), nitro,cyano, halo, OH, NH₂, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₃-C₆ cycloalkyl, C₄-C₇ cycloalkylalkyl, monocyclic aryl,monocyclic hetereoaryl, polycyclic aryl, and polycyclic heteroaryl.

The term “monocyclic” indicates a molecular structure having one ring.

The term “polycyclic” indicates a molecular structure having two or morerings, including, but not limited to, fused, bridged, or spiro rings.

The term “halogen” means fluorine, chlorine, bromine, or iodine.

The term “cyano” means a cyano group as shown below

The term “alkoxy” means groups of from 1 to 8 carbon atoms of astraight, branched, or cyclic configuration and combinations thereofattached to the parent structure through an oxygen. Examples includemethoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyloxy, andthe like. Lower-alkoxy refers to groups containing one to four carbons.For the purposes of the present patent application, alkoxy also includesmethylenedioxy and ethylenedioxy in which each oxygen atom is bonded tothe atom, chain, or ring from which the methylenedioxy or ethylenedioxygroup is pendant so as to form a ring. Thus, for example, phenylsubstituted by alkoxy may be, for example,

Monomers and compounds as described herein may contain one or moreasymmetric centers and may thus give rise to enantiomers, diastereomers,and other stereoisomeric forms. Each chiral center may be defined, interms of absolute stereochemistry, as (R)- or (S)-. The presentinvention is meant to include all such possible isomers, as well asmixtures thereof, including racemic and optically pure forms. Opticallyactive (R)- and (S)-, (−)- and (+)-, or (D)- and (L)-isomers may beprepared using chiral synthons or chiral reagents, or resolved usingconventional techniques. When the compounds described herein containolefinic double bonds or other centers of geometric asymmetry, andunless specified otherwise, it is intended that the compounds includeboth E and Z geometric isomers. Likewise, all tautomeric forms are alsointended to be included.

As referred to herein, a “polyvinyl alcohol containing unit” is a unitcomprising the chemical formula of CH₂═CH(OH) or

wherein

is the point of attachment of A to X and wherein CH₂═CH(OH) and

can be optionally substituted.

As used herein, a “saccharide containing unit” is any chemical unitcomprising a saccharide, in particular, a chemical unit comprising amonosaccharide, a disaccharide, or an oligosaccharide.

Monosaccharides are aldehydes or ketone derivatives of polyhydroxyalcohols having the general structure of (CH₂O)_(n), where n≥3. Themonosaccharide can be, without limitation, a substituted orunsubstituted triose, triulose, tetrose, tetulose, pentose, pentulose,penturonic acid, hexose, hexulose, hexuronic acid, heptose, heptulose,or hepturonic acid in its dextro (D-) or levo (L) form. Exemplarymonosaccharides include, without limitation, a substituted orunsubstituted, erythrose, threose, ribose, arabinose, xylose, lyxose,allose, altrose, glucose, mannose, gulose, idose, galactose, talose,fucose, fructose, erythrulose, ribulose, xylulose, psicose, sorbose,tagatose, and derivatives thereof, such as aldonic and uronic acids(e.g., gluconic acid, mannuronic acid, glucuronic acid, galacturonicacid, mannuronic acid, xyluronic acid), deoxy sugars (e.g., deoxyribose,rhamnose, and fucose), and amino sugars (e.g., glucosamine,galactosamine, N-acetylmuramic acid), the like. Other monosaccharidessuitable for use in the monomers and polymers as described herein arewell known in the art.

Disaccharides comprise two monosaccharides linked together by glycosidicbonds, and oligosaccharides comprise more than two, usually three to tenmonosaccharides linked together by glycosidic bonds. A disaccharide oroligosaccharide containing unit of the monomers and polymers asdescribed herein may comprise one type, or more than one type, ofmonosaccharide. Exemplary disaccharides include, without limitation,sucrose, lactose, maltose, trehalose, cellobiose, isomaltose, maltitoland the like. Exemplary oligosaccharides include, without limitation,fructo-oligosaccharides, galacto-oligosaccharides,gluco-oligosaccharides, raffinose, and the like.

In one embodiment, the saccharide containing unit of the monomerdescribed herein is mannuronate or guluronate selected from thefollowing:

where

is the point of attachment of A to X.

In one embodiment, the monomer of the present invention has the Formula(Ia):

In one embodiment, the monomer of the present invention has the Formula(Ib):

In another embodiment the monomer of the present invention has theFormula (Ic):

In another embodiment the monomer of the present invention has theformula selected from the group consisting of:

Another aspect of the present invention is directed to a polymer ofFormula (IV):

wherein

-   -   A is independently selected from a saccharide containing unit        and a polyvinyl alcohol containing unit for each monomer unit of        the polymer;    -   X is selected from the group consisting of O, NH, NR′, C(O), and        C₁₋₂₀ alkylene, wherein C₁₋₂₀ alkylene is optionally substituted        from 1 to 20 times with a substituent selected independently at        each occurrence thereof from the group consisting of OH,        halogen, cyano, —CF₃, and C₁₋₆ alkoxy.    -   Q is absent or is a linker;    -   Y is selected from the group consisting of

-   -   Z is selected from the group consisting of

-   -   m₁ is 0 to 50;    -   m₂ is 0 to 50;    -   m₃ is 0 to 50;    -   R is C₁₋₂₀ alkyl;    -   R′ is —C(O)—C₁₋₆ alkene;    -   R¹ is C₁₋₂₀ alkyl;    -   R² is C₁₋₂₀ alkyl;    -   R³ is C₁₋₂₀ alkyl,    -   k₁ is any integer; and    -   k₂ is independently selected for each monomer unit from 0 or 1,        with the proviso that at least one k₂ is 1;        wherein the monomer units of the polymer are the same or        different.

In one embodiment, Q is absent from the monomer unit of the polymer. Inanother embodiment, Q is present as a linker in the monomer unit of thepolymer. Q is an organic grouping containing any number of carbon atoms,1-30 carbon atoms, 1-20 carbon atoms, or 1-14 carbon atoms, andoptionally including one or more heteroatoms such as nitrogen, sulfur,or nitrogen in linear, branched, or cyclic structural formats.Representative Q groupings being C₁₋₂₀ alkylene, substituted C₁₋₂₀alkylene, phenoxy, substituted phenoxy, alkoxy, substituted alkoxy,C₃₋₂₀ cycloalkylene, substituted C₃₋₂₀ cycloalkylene, C₃₋₂₀cycloalkenylene, substituted C₃₋₂₀ cycloalkenylene, C₈₋₂₀cycloalkynylene, substituted C₈₋₂₀ cycloalkynylene, heterocyclylene,substituted heterocyclylene, arylene, substituted arylene, triazole,poly(ethylene glycol), or polypeptide moiety.

In one embodiment, Q is selected from the group consisting of C₁₋₂₀alkylene, C₃₋₂₀ cycloalkylene, arylene, heteroarylene, heterocyclene,—O—C₁₋₂₀ alkylene, poly(ethylene glycol), and polypeptide, wherein C₁₋₂₀alkylene, C₃₋₂₀ cycloalkylene, arylene, heteroarylene, heterocyclylene,or —O—C₁₋₂₀ alkylene is optionally substituted from 1 to 20 times with asubstituent selected independently at each occurrence thereof from thegroup consisting of —OH, halogen, cyano, —CF₃, C₁₋₆ alkyl, and C₁₋₆alkoxy, and wherein C₁₋₂₀ alkylene is optionally interrupted by one ormore heteroatoms selected from the group consisting of oxygen nitrogen,sulfur, or nitrogen.

In another embodiment, Q is heteroarylene.

In yet another embodiment, Q is

In one embodiment, the polymer of the present invention has Formula(IVa):

In another embodiment, the polymer of the present invention has Formula(IVb):

In another embodiment, the polymer of the present invention Formula(IVc):

A “polymer” as referred to herein, is a macromolecule comprising a setof regularly repeated chemical units, i.e., monomer units, that arejoined together to form a chain molecule. The repeated monomers can beof the same type, or of a limited number of different types. The numberof monomer units in the polymers as described herein can be any integer.In one embodiment, the number (k₁) of monomer units in the polymer isfrom about 5 to about 1,000 units, from about 5 to about 10,000 units,or from about 5 to about 100,000 units.

The monomers of the polymer may be joined together end-to-end, or in amore complicated fashion when forming the chemical chain. The polymersdescribed herein can be homopolymers, i.e., comprising only one type ofrepeating monomer unit, or copolymers, i.e., comprising more than onetype of repeating monomer unit. Copolymers of the present disclosureinclude alternating polymers, periodic polymers, random polymers, andblock polymers. The polymers described herein include linear polymersand branched polymers, including star polymers, brush polymers and combpolymers.

In one embodiment, the polymer as described herein is a polyvinylalcohol containing polymer (i.e., the polymer has a polyvinyl alcoholbackbone), where A at each instance of Formula IV is a polyvinyl alcoholcontaining unit.

As referred to herein, a “polyvinyl alcohol containing unit” is a unitcomprising the chemical formula of —CH₂CH(OH)— or

wherein

is the point of attachment of A to X and wherein —CH₂CH(OH)— and

can be optionally substituted.

In another embodiment, the polymer as described herein is a saccharidecontaining polymer (i.e., the polymer has a saccharide backbone). Inthis embodiment, A of Formula IV is a saccharide containing unit. Asdescribed supra, a saccharide containing unit is any chemical unitcomprising a saccharide, in particular, a chemical unit comprising amonosaccharide, a disaccharide, or an oligosaccharide. In accordancewith this embodiment, A is independently selected at each occurrencefrom a monosaccharide, disaccharide, and oligosaccharide. Exemplarysaccharides are described supra.

In one embodiment, the polymer as described herein is a homopolymercomprising one type of monosaccharide containing monomer unit asdescribed herein. In another embodiment, the homopolymer comprises onetype of disaccharide containing monomer unit as described herein. Inanother embodiment, the homopolymer comprises one type ofoligosaccharide containing monomer unit as described herein.

In another embodiment, the polymer is a copolymer containing two or moredifferent monomer units. In one embodiment the copolymer comprises twoor more different monosaccharide containing monomer units as describedherein. In another embodiment, the copolymer comprises two or moredifferent disaccharide containing monomer units. In another embodiment,the copolymer comprises two or more different oligosaccharide containingmonomer units. In another embodiment, the copolymer comprises two ormore different monomer units, where each monomer unit is independentlyselected from a monosaccharide, disaccharide, and oligosaccharidecontaining monomer unit.

In one embodiment, the polymer of the present invention is a modifiedsaccharide polymer. Saccharide polymers, also known as polysaccharidesor polymeric carbohydrates, are polymers comprising repeating monomericunits of one or more saccharides linked together by glycosidic linkages.Saccharide polymers are well known in the art, including, for exampleand without limitation, alginate, hyaluronic acid, chitin, cellulose,starch, agarose, dextran, carrageenan, guar, chondroitin, dermatan,among others. Thus, a modified saccharide polymer of the presentinvention is a polymer where A of Formula IV is a monomeric saccharideunit or a monosaccharide unit of a known saccharide polymer, and k₂ ofone or more monomeric units is 1.

Accordingly, in one embodiment, the polymer of the present invention isa modified cellulose polymer. According to this embodiment, A of FormulaIV comprises a glucose unit or substituted glucose unit to form themodified cellulose polymer or derivative thereof (e.g., a modified alkylcellulose, hydroxyalkyl cellulose, carboxyalkyl cellulose, or celluloseester).

In another embodiment, the polymer of the present invention is amodified dextran polymer. According to this embodiment, A of Formula IVis a D-glucopyranosyl unit to form the modified dextran polymer.

In another embodiment, the polymer of the present invention is amodified agarose polymer. According to this embodiment, A of Formula IValternates and is selected from D-galactose and3,6-anhydro-L-galactopyranose to form a modified agarose polymer.

In another embodiment, the polymer of the present invention is amodified chitosan polymer. According to the embodiment, A of Formula IVis independently selected from D-glucosamine and N-acetyl-D-glucosamineto form a modified chitosan polymer.

In another embodiment, the polymer of the present invention is amodified carrageenan polymer. According to this embodiment, A of FormulaIV alternates and is selected from galactose and 3,6 anhydrogalactose toform the modified carrageenan polymer.

In another embodiment, the polymer of the present invention is amodified hyaluronan polymer. According to this embodiment, A of FormulaIV alternates and is selected from glucuronic acid andN-acetyl-D-glucosamine to form a modified hyaluronan polymer.

In another embodiment, the polymer of the present invention is amodified chondroitin polymer. According to this embodiment, A of FormulaIV alternates and is selected from glucuronic acid andN-acetylgalactosamine to form the modified chondroitin sulfate polymer.

In another embodiment, the polymer of the present invention is amodified dermatan polymer. According to this embodiment, A of Formula IValternates and is selected from iduronic acid and N-acetylgalactosamineto form a modified dermatan sulfate polymer.

In another embodiment, the polymer of the present invention is a guarpolymer. According to this embodiment, A of Formula IV is independentlyselected from galactose and mannose to form a modified guar polymer.

In one embodiment, the polymer of the present disclosure is a modifiedalginate polymer. Alginate is a linear copolymer with homopolymericblocks of (1-4)-linked β-D-mannuronate and α-L-guluronate. In accordancewith this embodiment, A at each occurrence in Formula IV is selectedfrom β-D-mannuronate and α-L-guluronate. In one embodiment, the polymercomprises homopolymeric blocks of mannuronate containing monomer units.In another embodiment, the polymer comprises homopolymeric blocks ofguluronate containing monomer units. In another embodiment, the polymercomprises alternating mannuronate and guluronate containing monomerunits or alternating blocks of mannuronate and guluronate containingmonomer units. In one embodiment, the ratio of mannuronate to guluronatein the polymer of the present invention is about 1. In anotherembodiment, the ratio of mannuronate to guluronate is greater than 1. Inanother embodiment, the ratio of mannuronate to guluronate is less than1.

In accordance with this aspect of the present invention, exemplaryalginate polymers of the disclosure comprise monomer units where A isindependently selected at each occurrence from:

where

is the point of attachment of A to X.

Exemplary modified alginate polymers of the present invention includemonomeric units independently selected at each occurrence from thefollowing:

In accordance this aspect of the invention, k₂ of the polymer of FormulaIV is independently selected for each monomer unit from 0 or 1, with theproviso that at least one k₂ is 1. In one embodiment, k₂ is 1 for atleast 1% of the monomer units of the polymer as described herein. Inanother embodiment, k₂ is 1 for at least 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or moreof the monomer units of the polymer as described herein. In oneembodiment, k₂ is 1 for 100% of the monomer units of the polymer asdescribed herein.

Another aspect of the present invention relates to a polymer comprisingone or more monomers of Formula (I):

and further comprising

-   -   one or more monomers of Formula (II):

A-L¹-L²-L³-R⁴  (II), wherein

-   -   L¹ is selected from the group consisting of O, NH, NR′, C(O),        and C₁₋₂₀ alkylene, wherein C₁₋₂₀ alkylene is optionally        substituted from 1 to 20 times with a substituent selected        independently at each occurrence thereof from the group        consisting of OH, halogen, cyano, —CF₃, and C₁₋₆ alkoxy,    -   L² is absent or is C₁₋₂₀ alkylene, wherein C₁₋₂₀ alkylene is        optionally substituted from 1 to 20 times with a substituent        selected independently at each occurrence thereof from the group        consisting of OH, halogen, cyano, —CF₃, and C₁₋₆ alkoxy;    -   L³ is selected from the group consisting of C₁₋₂₀ alkylene,        C₁₋₂₀ alkenylene, C₃₋₁₂ cycloalkenylene, and arylene, wherein        arylene is optionally substituted from 1 to 3 times with a        substituent selected independently at each occurrence thereof        from the group consisting of heteroarylene and heterocyclylene;    -   R⁴ is selected from the group consisting of H, SH, N₃, C₁₋₆        alkyl, C₂₋₂₀ alkenyl, C₂₋₂₀ alkynyl, C₃₋₁₂ cycloalkenyl, C₃₋₁₂        cycloalkynyl, heteroaryl, and heterocyclyl, wherein C₁₋₆ alkyl,        C₂₋₂₀ alkenyl, C₂₋₂₀ alkynyl, C₃₋₁₂ cycloalkenyl, C₃₋₁₂        cycloalkynyl, heteroaryl, and heterocyclyl is optionally        substituted from 1 to 3 times with a substituent selected        independently at each occurrence thereof from the group        consisting of H, OH, halogen, cyano, —CF₃, and C₁₋₆ alkoxy.

In one embodiment of this aspect of the present invention, the polymerhas Formula (IIa):

-   -   wherein n₁, n₂, and n₃ are any integer.

In one embodiment, n₁, n₂, and n₃ are each selected independently froman integer of 1 to 10,000.

Exemplary polymers in accordance with this aspect of the disclosure,include, without limitation, the following polymers:

-   -   where n₁, n₂ and n₃ are any integer. In one embodiment, n₁, n₂,        and n₃ are each selected independently from an integer of 1 to        10,000.

Another aspect of the present invention relates to a monomer of Formula(III):

-   -   wherein    -   X¹ is absent or is

-   -   Y¹ is

-   -   Z¹ is

-   -   m₄ is 1 to 50;    -   m₅ is 0 to 10;    -   m₆ is 1 to 50;    -   R″ is H or C₁₋₆ alkyl;    -   R⁴ is C₁₋₂₀ alkyl;    -   R⁵ is C₁₋₂₀ alkyl; and    -   R⁶ is C₁-20 alkyl,    -   with the proviso that when X¹ is absent, Y¹ is not

In one embodiment, the monomer of Formula (III) has Formula (III′):

In one embodiment the monomer of Formula (III) has Formula (IIIa):

In one embodiment, the monomer of Formula (III) has Formula (IIIa′):

In another embodiment, the monomer of Formula (III) has Formula (IIb):

In another embodiment, the monomer of Formula (III) has Formula (IIIb′):

In another embodiment, the monomer of Formula (III) has Formula (IIc):

In another embodiment, the monomer of Formula (III) has Formula (IIIc′):

In another embodiment, an exemplary monomer of Formula (III) is selectedfrom the group consisting of:

Another aspect of the present invention relates to a polymeric networkof crosslinked monomers of Formula (III):

In one embodiment, the network is a homopolymeric network, comprisingone type of monomer of Formula (III). In another embodiment, the networkis heteropolymeric, comprising two or more different monomers of Formula(III).

In accordance with this embodiment, the polymeric network of monomers isformed using a crosslinker agent, and the monomers of the network arelinked together via the crosslinker. In one embodiment, the crosslinkingagent is a zwitterionic crosslinking agent. The zwitterioniccrosslinking agent can be copolymerized with the monomers andco-monomers of Formula III to provide crosslinked polymeric networks ofthe present invention. Suitable crosslinkers that can be used accordingto the present invention include bifunctional zwitterioniccarboxybetaine diacrylamide cross-linker (CBAAX), as well as any of thezwitterionic crosslinking agents disclosed in U.S. Patent ApplicationPublication No. 20170009069 to Jiang et al., which is herebyincorporated by reference in its entirety.

Another aspect of the present invention relates to a hydrogel comprisingany one or more of the polymers described herein (i.e., polymers ofFormula IV), any one or more monomers of Formula III, any one or more ofthe polymeric networks as described herein, or any combination thereof.Preferred hydrogels as described herein are zwitterionic hydrogels.

Hydrogels of the present invention can be homopolymeric hydrogels,copolymeric hydrogels, or multipolymeric hydrogels. In one embodiment,the hydrogel comprises one type of polymer as described herein. Inanother embodiment, the hydrogel comprises two or more different typesof polymers as described herein. In another embodiment, the hydrogel ismade from a single type of monomer of Formula III. In anotherembodiment, the hydrogel comprises two or more different types ofmonomers of Formula III. In another embodiment, the hydrogel is madefrom a polymeric network comprising a single type of monomer of FormulaIII. In another embodiment, the hydrogel is made from a polymericnetwork comprising one or more different types of monomers of FormulaIII.

In one embodiment, the hydrogel is a saccharide hydrogel, i.e., ahydrogel comprising saccharide containing polymers as described here. Inanother embodiment, the hydrogel is an alginate hydrogel, i.e., ahydrogel comprising modified alginate polymers as described herein.

Hydrogels of the present invention are formed using conventional methodsknown to those in the art and described in the Examples herein. Forexample the crosslinks between the polymers and monomers of the hydrogelcan be formed via chemical crosslinking reaction, ionizing radiation, orphysical interactions.

In one embodiment, the hydrogel is formed using click chemistry. Clickchemistry encompasses chemical reactions used to couple two compoundstogether which are high yielding, wide in scope, create only byproductsthat can be removed without chromatography, are stereospecific, simpleto perform, and can be conducted in easily removable or benign solvents.Examples of reactions which fulfill these criteria include thenucleophilic ring opening of epoxides and aziridines, non-aldol typecarbonyl reactions, including the formation of hydrazones andheterocycles, additions to carbon-carbon multiple bonds, includingMichael Additions, and cycloaddition reactions, such as a 1,3-dipolarcycloaddition reaction (i.e. a Huisgen cycloaddition reaction). Seee.g., Moses and Moorhouse, Chem Soc. Rev., 36:1249-1262 (2007); Kolb andSharpless, Drug Discovery Today, 8(24): 1128-1137 (2003); and Kolb etal., Angew. Chem. Int. Ed. 40:2004-2021 (2001), which are herebyincorporated by reference in their entirety.

In one embodiment the hydrogel is formed by the reaction between alkeneand tetrazine substituents of the polymers. In another embodiment thehydrogel is formed by the reaction between norbornene and tetrazinesubstituents of the polymers.

Hydrogels according to the present invention can be prepared accordingto the Schemes A and B.

Another aspect of the present invention relates to a capsule comprisingthe hydrogel of the present invention and a therapeutic agentencapsulated in the hydrogel.

A “capsule,” as used herein, refers to a particle having a mean diameterof about 150 μm to about 5 cm. In one embodiment, the capsules of thepresent invention have a mean diameter of about 150 μm to about 8 mm. A“microcapsule” as referred to herein has a mean diameter of about 150 μmto about 1000 μm. The capsule or microcapsule is formed of the polymers,polymeric matrix, or hydrogels as described herein. The capsule maycomprise a cross-linked hydrogel core that is surrounded by one or morepolymeric shells, one or more cross-linked hydrogel layers, across-linked hydrogel coating, or a combination thereof. The capsule canbe any suitable shape for cell encapsulation or encapsulation of atherapeutic agent. Useful shapes include spheres, sphere-like shapes,spheroids, spheroid-like shapes, ellipsoids, ellipsoid-like shapes,stadiumoids, stadiumoid-like shapes, disks, disk-like shapes, cylinders,cylinder-like shapes, rods, rod-like shapes, cubes, cube-like shapes,cuboids, cuboid-like shapes, toruses, torus-like shapes, and flat andcurved surfaces.

When used for cell encapsulation, the capsule may contain one or morecells dispersed in the cross-linked hydrogel, thereby “encapsulating”the cells. The rate of molecules entering the capsule necessary for cellviability and the rate of therapeutic products and waste materialexiting the capsule membrane can be selected by modulating capsulepermeability. Capsule permeability can also be modified to limit entryof immune cells, antibodies, and cytokines into the capsule. Generally,known methods of forming hydrogel capsules can produce capsules havinglimited entry of immune cells, antibodies, and cytokines into thecapsule. Since different cell types have different metabolicrequirements, the permeability of the capsule can be optimized based onthe cell type encapsulated in the hydrogel. The diameter of the capsulesis an important factor that influences both the immune response towardsthe cell capsules as well as the mass transport across the capsulemembrane.

The therapeutic agent can be any biologically reactive agent including,for example, and without limitation, therapeutic proteins, peptides,antibodies or fragments thereof, antibody mimetics, and other bindingmolecules, nucleic acids, small molecules, hormones, growth factors,angiogenic factors, cytokines, and anti-inflammatory agents.

The types of drugs (or therapeutic agents) that can be delivered usingthe capsules and microcapsules as described herein are numerous, andinclude both small molecular weight compounds in the size range from 100daltons to about 1,000 daltons as well as the larger macromoleculardrugs, such as peptide and protein drugs in the size range from about1,000 daltons to about 100,000 daltons, and beyond. The capsules made ofthe polymers and hydrogels described herein are particularly well suitedto deliver drugs having relatively low effective doses, e.g., in themicrograms/day, nanograms/day, and even picograms/day range.

Protein and/or peptide therapeutic agents which may be contained withinthe capsule for delivery upon implantation in a subject include, withoutlimitation, peptide hormones such as insulin, glucagon, parathyroidhormone, calcitonin, vasopression, renin, prolactin, growth hormone, thegonadotropins, including chorionic gonadotropin, follicle stimulatinghormone, thyroid stimulating hormone, and luteinizing hormone;physiologically active enzymes such as transferases, hydrolases, lyases,isomerases, phosphatases, glycosidases, superoxide dismutase, factorVIII, plasminogen activators; and other therapeutic agents includingprotein factors such as epidermal growth factor, insulin-like growthfactor, tumour necrosis factor, transforming growth factors, fibroblastgrowth factors, platelet-derived growth factors, erythropoietin, colonystimulating factors, bone morphogenetic proteins, interleukins, andinterferons. Non-protein macromolecules, particularly includingpolysaccharides, nucleic acid polymers, and therapeutic secondarymetabolites, including plant products such as vinblastine, vincristine,taxol, and the like may also be delivered using the present system.Small molecular weight compounds may also be delivered.

In one embodiment, the therapeutic agent is a biological agent producedand/or secreted or released from tissue and/or a preparation of cellsencapsulated within or residing within the capsule as described herein.The cells may comprise naturally occurring or genetically engineeredcells which may be in the form of single cells and/or cell clusters. Inone embodiment, the cells within the capsule secrete one or morebiological factors that are useful in the treatment of a disease orcondition. These factors are secreted from the cells, released from thehydrogel or polymeric layer of the capsule, and are delivered to ordiffuse to surrounding target cells, tissue, or organ in need thereof.Suitable cells include, without limitation, one or more cell typesselected from the group consisting of smooth muscle cells, cardiacmyocytes, platelets, epithelial cells, endothelial cells, urothelialcells, fibroblasts, embryonic fibroblasts, myoblasts, chondrocytes,chondroblasts, osteoblasts, osteoclasts, keratinocytes, hepatocytes,bile duct cells, pancreatic islet cells, thyroid, parathyroid, adrenal,hypothalamic, pituitary, ovarian, testicular, salivary gland cells,adipocytes, stem cells, including mesenchymal stem cells, neural cells,endothelial progenitor cells, hematopoietic cells, embryonic stem cells,induced pluripotent stem cells, or cells derived from such stem cells.Suitable cells include xenogeneic, autologous, or allogeneic cells.Cells can be primary cells or cells derived from the culture andexpansion of a cell obtained from a subject. Cells can also beimmortalized cells.

In one embodiment, the cells are insulin secreting cells. An insulinsecreting cells is a cell that produces insulin, preferably in responseto glucose levels. Insulin secreting cells include pancreatic isletcells, insulin-producing cells derived from stem cells, and cellsgenetically engineered to produce insulin. In one embodiment, the cellsare mammalian insulin secreting cells. In one embodiment, the cells arehuman insulin secreting cells. In one embodiment, the cells are humanpancreatic islet cells. Islet cells are endocrine cells derived from amammalian pancreas. Islet cells include alpha cells that secreteglucagon, beta cells that secrete insulin and amylin, delta cells thatsecrete somatostatin, PP cells that secrete pancreatic polypeptide, orepsilon cells that secrete ghrelin. The term includes homogenous andheterogeneous populations of these cells. In preferred embodiments, apopulation of islet cells contains at least beta cells.

Methods of isolating pancreatic islet cells that are suitable forencapsulation in the capsule as described herein are known in the art.See e.g. Field et al., Transplantation 61:1554 (1996); Linetsky et al.,Diabetes 46:1120 (1997), which are incorporated by reference in theirentirety). Fresh pancreatic tissue can be divided by mincing, teasing,comminution and/or collagenase digestion. The islets can then beisolated from contaminating cells and materials by washing, filtering,centrifuging or picking procedures. Methods and apparatus for isolatingand purifying islet cells are described in U.S. Pat. No. 5,447,863 toLangley, U.S. Pat. No. 5,322,790 to Scharp et al., U.S. Pat. No.5,273,904 to Langley, and U.S. Pat. No. 4,868,121 to Scharp et al.,which are hereby incorporated by reference in their entirety. Theisolated pancreatic cells may optionally be cultured prior to inclusionin the hydrogel capsule using any suitable method of culturing isletcells as is known in the art. See e.g., U.S. Pat. No. 5,821,121 toBrothers which is hereby incorporated by reference in its entirety.Isolated cells may be cultured in a medium under conditions that helpsto eliminate antigenic components. Insulin-producing cells can also bederived from stem cells and cell lines and can be cells geneticallyengineered to produce insulin. See e.g., U.S. Patent ApplicationPublication No. 20040005301 to Goldman and U.S. Pat. No. 9,624,472 toFirpo, which are hereby incorporated by reference in their entirety.

As noted above, suitable cells include progenitor and/or stem cells.Suitable stem cells may be pluripotent, multipotent, oligopotent, orunipotent cells or cell populations, and include embryonic stem cells,epiblast cells, primitive ectoderm cells, and primordial germ cells. Inanother embodiment, suitable stem cells also include induced pluripotentstem (iPS) cells, which are pluripotent stem cells derived from anon-pluripotent cell. See Zhou et al., Cell Stem Cell 4:381-384 (2009);Yu et al., Science 324(5928):797-801 (2009); Yu et al., Science318(5858):1917-20 (2007); Takahashi et al., Cell 131:861-72 (2007); andTakahashi and Yamanaka, Cell 126:663-76 (2006), which are herebyincorporated by reference in their entirety. In accordance with thisembodiment, the capsule may further comprise the growth anddifferentiation factors suitable for promoting stem cell differentiationinto a desired population of cells capable of producing and releasingthe therapeutic agent of interest.

Suitable cells for encapsulation in the capsule described herein can bederived from any animal capable of generating the desired cells. Theanimals from which the cells are harvested may be vertebrate orinvertebrate, mammalian or non-mammalian, human or non-human. Examplesof animal sources include, but are not limited to, primate, rodent,canine, feline, equine, bovine, or porcine. The cells may be obtainedfrom or comprise a primary cell preparation or immortalized cellspreparations. The encapsulated cells may be isolated from the samespecies as the implant recipient or from a different species than theimplant recipient.

In some embodiments, the capsule described herein comprises a celldensity between approximately 1×10⁵ or 1×10⁶ cells/ml to about 1×10¹⁰cells/mL or more. In one embodiment, the cell holding capacity of thecapsule is based, at least in part, on the size of the capsule. In oneembodiment, the capsule membrane prohibits cells in the hydrogel fromescaping the capsule. The cells are capable of surviving in vivo in thecapsule for at least a month, two months, three months, four months,five months, six months, seven months, eight months, nine months, tenmonths, eleven months, twelve months or a year or more with afunctionality that represents at least about 50%, 55%, 60%, 65%, 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the function expressed atthe time the cells are/were introduced into the capsule or at the timethe cells fully develop and/or mature in the capsule, e.g., implantationof progenitor cells which need to further develop or mature tofunctional cells in vivo. In some embodiments, the cells or cellpreparation in the system expand within the capsule to increase celldensity and/or cell function upon implantation of the system in vivo.

When the capsule contains cells or a cell preparation, additional cellspecific growth and/or differentiation factors may be added to thehydrogel or polymeric solution to enhance cell growth, differentiation,and survival. These factors include supplements (e.g., glutamine,non-essential amino acids), growth factors (e.g., epidermal growthfactors, fibroblast growth factors, transforming growth factor/bonemorphogenetic proteins, platelet derived growth factors, insulin growthfactors, cytokines), extracellular matrix proteins (e.g., fibronectin,laminin, heparin, collagen, glycosaminoglycan, proteoglycan, elastin,chitin derivatives, fibrin, and fibrinogen), angiogenic factors (e.g.,FGF, bFGF, acid FGF (aFGF), FGF-2, FGF-4, EGF, PDGF, TGF-beta,angiopoietin-1, angiopoietin-2, placental growth factor (PlGF), VEGF,and PMA (phorbol 12-myristate 13-acetate)), and signaling factors and/ortranscription factors.

Another aspect of the present invention is directed to a method ofdelivering a therapeutic agent to a subject. This method involvesadministering the capsule comprising the hydrogel and a therapeuticagent encapsulated by the hydrogel to the subject.

The capsule described herein can be employed for treating a variety ofdiseases and conditions requiring a continuous supply of biologicallyactive substance or substances to the subject. The capsule may containhomogenous or heterogenous mixtures of biologically active agents and/orcells, or cells producing one or more biologically active substances ofinterest. The biologically active agents and/or cells are whollyencapsulated within the hydrogel layer of the capsule. Such asemi-permeable outer layer allows the encapsulated biologically activesubstance of interest (e.g., insulin, glucagon, pancreatic polypeptide,and the like in the case of treating diabetes) to pass out of thesystem, making the active substance available to target cells outsidethe system and in the recipient subject's body. In one embodiment, thecapsule membrane is a semi-permeable membrane that allows nutrientsnaturally present in the subject to pass through the membrane to provideessential nutrients to cells present in the hydrogel. At the same time,such a semi-permeable membrane prevents the recipient subject's cells,more particularly, their immune system cells, from passing through andinto the capsule to harm the cells in the system. For example, in thecase of diabetes, this approach can allow glucose and oxygen (e.g.,contained within the body) to stimulate insulin-producing cells of thecapsule to release insulin as required by the body in real time whilepreventing host immune system cells from recognizing and destroying theimplanted cells.

Another aspect of the present invention is directed to a method oftreating a diabetic subject. This method involves selecting a subjecthaving diabetes, and implanting the capsule comprising the hydrogel anda therapeutic agent encapsulated by the hydrogel to the subject. Inaccordance with this embodiment, the capsule contains a preparation ofinsulin secreting cells encapsulated in the hydrogel, and thetherapeutic agent delivered to the subject is insulin.

In accordance with one embodiment of this aspect of the invention, thesubject having diabetes has type-1 diabetes (also called juvenilediabetes). In another embodiment, the subject has type-2 diabetes. Inanother embodiment, the subject has Maturity Onset Diabetes of the Young(MODY).

The capsule can be surgically implanted into subjects. In oneembodiment, the capsule is implanted using minimally invasive surgicaltechniques such as laparoscopy. The capsule can be implantedpercutaneously, subcutaneously, intraperitoneally, intrathoracically,intramuscularly, intraarticularly, intraocularly, or intracerebrallydepending on the therapeutic agent being delivered, condition to betreated, and tissue or organ targeted for delivery.

Subjects amenable to treatment with the capsule according to the presentinvention include mammals (e.g., a human, horse, pig, rabbit, dog,sheep, goat, non-human primate, cow, cat, guinea pig or rodent), fish,birds, reptiles, and amphibians. The term does not denote a particularage or sex. Thus, adult and newborn subjects, as well as fetuses,whether male or female, are intended to be covered. In some embodiments,the subject is afflicted with a disease or disorder. The term “subject”includes human and veterinary subjects.

Another aspect of the present invention is directed to surfaces coatedwith the polymers, polymeric networks, and hydrogels described herein.The polymers, polymeric networks, and hydrogels can be advantageouslyused to coat surfaces of a variety of devices including, for example,medical devices to provide biocompatible, non-fibrotic, and non-foulingsurfaces. Accordingly, in another aspect, the invention provides devicesand materials having a surface (i.e., one or more surfaces) to whichhave been applied (e.g., coated, covalently coupled, ionicallyassociated, hydrophobically associated) one or more polymers, polymericnetworks, or hydrogels of the invention. Representative devices andcarriers that may be advantageously treated or coated with the polymersand polymeric networks described herein include: particles (e.g.,nanoparticles) having a surface treated with, modified to include, orincorporating the polymers or polymeric network described herein; a drugcarrier having a surface treated with, modified to include, orincorporating the polymers or polymeric network described herein; anon-viral gene delivery system having a surface treated with, modifiedto include, or incorporating the polymers or polymeric network describedherein; a biosensor having a surface treated with, modified to include,or incorporating the polymers or polymeric network described herein;devices for bioprocesses or bioseparations, such as membranes formicrobial suspension, hormone separation, protein fractionation, cellseparation, waste water treatment, oligosaccharide bioreactors, proteinultrafiltration, and dairy processing having a surface treated with,modified to include, or incorporating the polymers or polymeric networkdescribed herein; implantable sensor having a surface treated with,modified to include, or incorporating the polymers or polymeric networkdescribed herein; subcutaneous sensor having a surface treated with,modified to include, or incorporating the polymers or polymeric networkdescribed herein; an implant, such as a breast implant, cochlearimplant, and dental implant having a surface treated with, modified toinclude, or incorporating the polymers or polymeric network describedherein; a tissue scaffold having a surface treated with, modified toinclude, or incorporating the polymers or polymeric network describedherein; implantable medical devices, such as an artificial joint,artificial heart valve, artificial blood vessel, pacemaker, leftventricular assist device (LVAD), artery graft, and stent having asurface treated with, modified to include, or incorporating the polymersor polymeric network described herein; and medical devices, such as anear drainage tube, feeding tube, glaucoma drainage tube, hydrocephalousshunt, keratoprosthesis, nerve guidance tube, urinary catheter, tissueadhesive, and x-ray guide having a surface treated with, modified toinclude, or incorporating the polymers or polymeric network describedherein.

The capsules, products, devices, and surfaces made from or coated withthe polymers or polymeric network as described herein can remainsubstantially free of fibrotic effects, or can continue to exhibit areduced foreign body response, for 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 2 months, 3 months, 4months, 5 months, 6 months, 7 months, 8, months, 9 months, 10 months, 11months, 1 year, 2 years, or longer after administration or implantationin a subject.

Methods of Synthesis

Another aspect of the present invention relates to methods of preparingthe monomers and polymers as described herein.

Accordingly, the polymers comprising one or more monomers of Formula (I)can be prepared according to Schemes 1-2 outlined below.

M is metal

Reaction between the saccharide salt (1) and compound (2) leads toformation of the saccharide polymers comprising one or more covalentlymodified monomers of Formula (I).

Amine (3) can be protected using a suitable protecting group. Couplingof the protected amine (4) with the compound (5) leads to formation ofthe compound (6). The coupling reaction can be carried out in a varietyof solvents, for example in acetonitrile, methanol, ethanol, methylenechloride (CH₂Cl₂), tetrahydrofuran (THF), dimethylformamide (DMF), orother such solvents or in the mixture of such solvents. During thecoupling process, the non-participating carboxylic acids on the compound5 can be protected by a suitable protecting group which can beselectively removed at a later time if desired. A detailed descriptionof these groups and their selection and chemistry is contained in “ThePeptides, Vol 3”, Gross and Meinenhofer, Eds., Academic Press, New York,1981, which is hereby incorporated by reference in its entirety. Thus,useful protective groups for the amino group are benzyloxycarbonyl(Cbz), t-butyloxycarbonyl (t-BOC), 2,2,2-trichloroethoxycarbonyl (Troc),t-amyloxycarbonyl, 4-methoxybenzyloxycarbonyl,2-(trichlorosilyl)ethoxycarbonyl, 9-fluorenylmethoxy carbonyl (Fmoc),phthaloyl, acetyl (Ac), formyl, trifluoroacetyl, and the like. Removalof the protecting groups leads to formation of compound (2a).

Another aspect of the present invention relates to methods ofsynthesizing monomers of Formula (III). These monomers can be preparedaccording to Schemes 3-5 outlined below.

Reaction between compound (7) and sodium azide leads to formation ofazide (8). The reaction can be carried out in a variety of solvents, forexample in dimethyl sulfoxide (DMSO), methanol, ethanol,dimethylformamide (DMF), or other such solvents or in the mixture ofsuch solvents. Reaction between the azide (8) and the compound (5a)leads to formation of the compound (9). The reaction can be carried outin a variety of solvents, for example in acetonitrile, methanol,ethanol, methylene chloride (CH₂Cl₂), tetrahydrofuran (THF),dimethylformamide (DMF), or other such solvents or in the mixture ofsuch solvents. During this process, the non-participating carboxylicacids on the compound 5a can be protected by a suitable protecting groupwhich can be selectively removed at a later time if desired. A detaileddescription of these groups and their selection and chemistry iscontained in “The Peptides, Vol. 3”, Gross and Meinenhofer, Eds.,Academic Press, New York, 1981, which is hereby incorporated byreference in its entirety. Thus, useful protective groups for the aminogroup are benzyloxycarbonyl (Cbz), t-butyloxycarbonyl (t-BOC),2,2,2-trichloroethoxycarbonyl (Troc), t-amyloxycarbonyl,4-methoxybenzyloxycarbonyl, 2-(trichlorosilyl)ethoxycarbonyl,9-fluorenylmethoxycarbonyl (Fmoc), phthaloyl, acetyl (Ac), formyl,trifluoroacetyl, and the like.

Reaction between azide (9) and alkyne (10) leads to formation oftriazole (11). The reaction can be carried out in a variety of solvents,for example in dimethyl sulfoxide (DMSO), methanol, ethanol,dimethylformamide (DMF), or other such solvents or in the mixture ofsuch solvents. Removal of the protecting groups leads to formation ofcompound (IIIa″).

Reaction between the azide (8) and alkyne (10) leads to formation of thetriazole (12). The reaction can be carried out in a variety of solvents,for example in acetonitrile, methanol, ethanol, methylene chloride(CH₂Cl₂), tetrahydrofuran (THF), dimethylformamide (DMF), or other suchsolvents or in the mixture of such solvents. Reaction between the azide(12) and the compound (Sb) leads to formation of the monomer (IIIb′).The reaction can be carried out in a variety of solvents, for example inacetonitrile, methanol, ethanol, methylene chloride (CH₂Cl₂),tetrahydrofuran (THF), dimethylformamide (DMF), or other such solventsor in the mixture of such solvents.

Alkyne (10) can be prepared by reacting compound (13) with alkyne (14)in the presence of a base. This reaction can be carried out in a varietyof solvents, for example in acetonitrile, methanol, ethanol, methylenechloride (CH₂Cl₂), tetrahydrofuran (THF), dimethylformamide (DMF), orother such solvents or in the mixture of such solvents. Reaction betweenthe alkyne (10) and azide (15) leads to formation of the triazole (16).During this process, the non-participating carboxylic acids on thecompound 5a can be protected by a suitable protecting group which can beselectively removed at a later time if desired. A detailed descriptionof these groups and their selection and chemistry is contained in “ThePeptides, Vol 3”, Gross and Meinenhofer, Eds., Academic Press, New York,1981, which is hereby incorporated by reference in its entirety. Thus,useful protective groups for the amino group are benzyloxycarbonyl(Cbz), t-butyloxycarbonyl (t-BOC), 2,2,2-trichloroethoxycarbonyl (Troc),t-amyloxycarbonyl, 4-methoxybenzyloxycarbonyl,2-(trichlorosilyl)ethoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc),phthaloyl, acetyl (Ac), formyl, trifluoroacetyl, and the like. Thereaction can be carried out in a variety of solvents, for example inacetonitrile, methanol, ethanol, methylene chloride (CH₂Cl₂),tetrahydrofuran (THF), dimethylformamide (DMF), or other such solventsor in the mixture of such solvents. During this process, thenon-participating carboxylic acids on the compound 15 can be protectedby a suitable protecting group which can be selectively removed at alater time if desired. A detailed description of these groups and theirselection and chemistry is contained in “The Peptides, Vol. 3”, Grossand Meinenhofer, Eds., Academic Press, New York, 1981, which is herebyincorporated by reference in its entirety. Thus, useful protectivegroups for the amino group are benzyloxycarbonyl (Cbz),t-butyloxycarbonyl (t-BOC), 2,2,2-trichloroethoxycarbonyl (Troc),t-amyloxycarbonyl, 4-methoxybenzyloxycarbonyl,2-(trichlorosilyl)ethoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc),phthaloyl, acetyl (Ac), formyl, trifluoroacetyl, and the like. Azide(12) can be alkylated with alkyl halide (R⁴Hal) to form the compound(17). The reaction can be carried out in a variety of solvents, forexample in acetonitrile, methanol, ethanol, methylene chloride (CH₂Cl₂),tetrahydrofuran (THF), dimethylformamide (DMF), or other such solventsor in the mixture of such solvents. Removal of the protecting groupsleads to formation of monomer (IIIc′).

Another aspect of the invention relates to a process for preparation ofa monomer of Formula (IIIa′):

where

-   -   m₄ is 1 to 50;    -   m₅ is 0 to 10;    -   m₆ is 1 to 50;    -   R⁵ is C₁₋₂₀ alkyl; and    -   R⁶ is C₁₋₂₀ alkyl.

This process includes:

-   -   providing a compound of Formula (V):

where

-   -   PG is a suitable protecting group; and    -   forming the monomer of Formula (IIIa′) from compound of Formula        (V).

Any suitable protecting group (PG) can be used. A detailed descriptionof these groups and their selection and chemistry is contained in “ThePeptides, Vol. 3”, Gross and Meinenhofer, Eds., Academic Press, NewYork, 1981, which is hereby incorporated by reference in its entirety.

In one embodiment, PG is selected form the group consisting oft-butyloxycarbonyl (t-BOC), 9-fluorenylmethoxycarbonyl (Fmoc),benzyloxycarbonyl (Cbz), 2,2,2-trichloroethoxycarbonyl (Troc),t-amyloxycarbonyl, 4-methoxybenzyloxycarbonyl,2-(trichlorosilyl)ethoxycarbonyl, phthaloyl, acetyl (Ac), formyl, andtrifluoroacetyl.

One embodiment relates to a process for preparation of a monomer ofFormula (IIIa′), wherein said forming the monomer of Formula (IIIa′)comprises reacting the compound of Formula (V) with a suitableprotecting group removing agent.

Another embodiment relates to a process for preparation of a monomer ofFormula (IIIa′) that further includes:

-   -   providing a compound of Formula (VI):

-   -   and    -   forming the compound of Formula (V) from the compound of Formula        (VI).

A further embodiment relates to a process for preparation of a monomerof Formula (IIIa′), wherein said forming the compound of Formula (V)comprises reacting the compound of Formula (VI) with the compound ofFormula (VII):

under conditions effective to produce the compound of Formula (V).

Another embodiment relates to a process for preparation of a monomer ofFormula (IIIa′) that further includes: providing a compound of Formula(VIII):

-   -   and    -   forming the compound of Formula (VI) from the compound of        Formula (VIII).

A further embodiment relates to a process for preparation of a monomerof Formula (IIIa′), wherein said forming the compound of Formula (VI)comprises reacting the compound of Formula (VIII) with the compound ofFormula (IX):

-   -   wherein Hal is halogen,    -   under conditions effective to produce the compound of Formula        (VI).

Yet another embodiment relates to a process for preparation of a monomerof Formula (IIIa′) that further includes:

-   -   providing a compound of Formula (X):

-   -   and    -   forming the compound of Formula (VIII) from the compound of        Formula (X).

A further embodiment relates to a process for preparation of a monomerof Formula (IIIa′), wherein said forming the compound of Formula (VIII)comprises reacting the compound of Formula (X) with metal azide (MN₃),wherein M is any suitable metal, under conditions effective to producethe compound of Formula (VIII).

Yet another embodiment relates to a process for preparation of a monomerof Formula (IIIa′) that further includes: providing a compound ofFormula (XI):

-   -   and    -   forming the compound of Formula (VII) from the compound of        Formula (XI).

A further embodiment relates to a process for preparation of a monomerof Formula (IIIa′), wherein said forming the compound of Formula (VII)comprises reacting the compound of Formula (XI) with the compound ofFormula (XII):

-   -   under conditions effective to produce the compound of Formula        (VII).

Another aspect of the invention relates to a process for preparation ofa monomer of Formula (IIIb′):

where

-   -   m₄ is 1 to 50;    -   m₅ is 0 to 10;    -   m₆ is 1 to 50;    -   R⁵ is C₁₋₂₀ alkyl; and    -   R⁶ is C₁₋₂₀ alkyl.

This process includes:

-   -   providing a compound of Formula (XIII):

and

-   -   forming the monomer of Formula (IIIb′) from compound of Formula        (XIII).

One embodiment relates to a process for preparation of a monomer ofFormula (IIIb′), wherein said forming the monomer of Formula (IIIb′)comprises reacting the compound of Formula (XIII) with a compound ofFormula (XIV):

-   -   under conditions effective to produce the monomer of Formula        (IIIb′).

Another embodiment relates to a process for preparation of a monomer ofFormula (IIIb′) that further includes: providing a compound of Formula(XV):

-   -   and    -   forming the compound of Formula (XIII) from the compound of        Formula (XV).

A further embodiment relates to a process for preparation of a monomerof Formula (IIIb′), wherein said forming the compound of Formula (XIII)comprises reacting the compound of Formula (XV) with the compound ofFormula (VII):

under conditions effective to produce the compound of Formula (XIII).

Yet another embodiment relates to a process for preparation of a monomerof Formula (IIIb′) that further includes:

-   -   providing a compound of Formula (XI):

-   -   and    -   forming the compound of Formula (VII) from the compound of        Formula (XI).

A further embodiment relates to a process for preparation of a monomerof Formula (IIIb′), wherein said forming the compound of Formula (VII)comprises reacting the compound of Formula (XI) with the compound ofFormula (XII):

-   -   under conditions effective to produce the compound of Formula        (VII).

Another aspect of the invention relates to a process for preparation ofa monomer of Formula (IIIc′):

where

-   -   m₄ is 1 to 50;    -   m₆ is 1 to 50;    -   R⁴ is C₁₋₂₀ alkyl;

This process includes:

-   -   providing a compound of Formula (XVI):

and where

-   -   PG is a suitable protecting group; and    -   forming the monomer of Formula (IIIc′) from compound of Formula        (XVI).

Any suitable protecting group (PG) can be used. A detailed descriptionof these groups and their selection and chemistry is contained in “ThePeptides, Vol. 3”, Gross and Meinenhofer, Eds., Academic Press, NewYork, 1981, which is hereby incorporated by reference in its entirety.

In one embodiment, PG is selected form the group consisting oft-butyloxycarbonyl (t-BOC), 9-fluorenylmethoxycarbonyl (Fmoc),benzyloxycarbonyl (Cbz), 2,2,2-trichloroethoxycarbonyl (Troc),t-amyloxycarbonyl, 4-methoxybenzyloxycarbonyl,2-(trichlorosilyl)ethoxycarbonyl, phthaloyl, acetyl (Ac), formyl, andtrifluoroacetyl.

One embodiment relates to a process for preparation of a monomer ofFormula (IIIc′), wherein said forming the monomer of Formula (IIIc′)comprises reacting the compound of Formula (XVI) with a suitableprotecting group removing agent.

Another embodiment relates to a process for preparation of a monomer ofFormula (IIIc′) that further includes:

-   -   providing a compound of Formula (XVII):

-   -   and    -   forming the compound of Formula (XVI) from the compound of        Formula (XVII).

A further embodiment relates to a process for preparation of a monomerof Formula (IIIc′), wherein said forming the compound of Formula (XVI)comprises reacting the compound of Formula (XVII) with the compound ofFormula (XVIII):

R⁴Hal  (XVIII),

-   -   under conditions effective to produce the compound of Formula        (XVI).

Another embodiment relates to a process for preparation of a monomer ofFormula (IIIc′) that further includes:

-   -   providing a compound of Formula (VII):

-   -   and    -   forming the compound of Formula (XVII) from the compound of        Formula (VII).

A further embodiment relates to a process for preparation of a monomerof Formula (IIIc′), wherein said forming the compound of Formula (XVII)comprises reacting the compound of Formula (VII) with the compound ofFormula (XIX):

under conditions effective to produce the compound of Formula (XVII).

Yet another embodiment relates to a process for preparation of a monomerof Formula (IIIc′) that further includes:

-   -   providing a compound of Formula (XI):

-   -   and    -   forming the compound of Formula (VII) from the compound of        Formula (XI).

A further embodiment relates to a process for preparation of a monomerof Formula (IIIc′), wherein said forming the compound of Formula (VII)comprises reacting the compound of Formula (XI) with the compound ofFormula (XII):

-   -   under conditions effective to produce the compound of Formula        (VII).

Another aspect of the invention relates to a process for preparation ofa compound of Formula (XXa):

wherein

-   -   m₁ is 1 to 50;    -   m₂ is 1 to 10;    -   R₁ is C₁₋₂₀ alkyl; and    -   R₂ is C₁₋₂₀ alkyl.

This process includes:

-   -   providing a compound of Formula (XXI):

-   -   wherein        -   PG is a suitable protecting group; and        -   forming the compound of Formula (XXa) from compound of            Formula (XXI).

Any suitable protecting group (PG) can be used. A detailed descriptionof these groups and their selection and chemistry is contained in “ThePeptides, Vol. 3”, Gross and Meinenhofer, Eds., Academic Press, NewYork, 1981, which is hereby incorporated by reference in its entirety.

In one embodiment, PG is selected form the group consisting oft-butyloxycarbonyl (t-BOC), 9-fluorenylmethoxycarbonyl (Fmoc),benzyloxycarbonyl (Cbz), 2,2,2-trichloroethoxycarbonyl (Troc),t-amyloxycarbonyl, 4-methoxybenzyloxycarbonyl,2-(trichlorosilyl)ethoxycarbonyl, phthaloyl, acetyl (Ac), formyl, andtrifluoroacetyl.

One embodiment relates to a process for preparation of a compound ofFormula (XXa), wherein said forming the compound of Formula (XXa)comprises reacting the compound of Formula (XXI) with a suitableprotecting group removing agent.

Another embodiment relates to a process for preparation of a compound ofFormula (XXa) that further includes:

-   -   providing a compound of Formula (XXII):

-   -   and    -   forming the compound of Formula (XXI) from the compound of        Formula (XXII).

A further embodiment relates to a process for preparation of a compoundof Formula (XXa), wherein said forming the compound of Formula (XXI)comprises reacting the compound of Formula (XXII) with the compound ofFormula (XXIII):

-   -   under conditions effective to produce the compound of Formula        (XXI).

Yet another embodiment relates to a process for preparation of acompound of Formula (XXa) that further includes:

-   -   providing a compound of Formula (XXIV):

-   -   and    -   forming the compound of Formula (XXII) from the compound of        Formula (XXIV).

A further embodiment relates to a process for preparation of a compoundof Formula (XXa), wherein said forming the compound of Formula (XXII)comprises reacting the compound of Formula (XXIV) with a suitableprotecting group introducing agent, under conditions effective toproduce the compound of Formula (XXII).

Another aspect of the invention relates to a process for preparation ofa compound of Formula (XXb):

wherein

-   -   m₁ is 1 to 50;    -   m₂ is 1 to 10;    -   R₁ is C₁₋₂₀ alkyl;    -   R₂ is C₁₋₂₀ alkyl.

This process includes:

-   -   providing a compound of Formula (XXV):

wherein

-   -   PG* is a suitable protecting group;    -   R₄ is C₁₋₆ alkyl, and    -   forming the compound of Formula (XXb) from compound of Formula        (XXV).

Any suitable protecting group (PG*) can be used. A detailed descriptionof these groups and their selection and chemistry is contained in “ThePeptides, Vol. 3”, Gross and Meinenhofer, Eds., Academic Press, NewYork, 1981, which is hereby incorporated by reference in its entirety.

In one embodiment PG* is selected form the group consisting oft-butyloxycarbonyl (t-BOC), 9-fluorenylmethoxycarbonyl (Fmoc),benzyloxycarbonyl (Cbz), 2,2,2-trichloroethoxycarbonyl (Troc),t-amyloxycarbonyl, 4-methoxybenzyloxycarbonyl,2-(trichlorosilyl)ethoxycarbonyl, phthaloyl, acetyl (Ac), formyl, andtrifluoroacetyl.

Another embodiment relates to a process for preparation of a compound ofFormula (XXb), wherein said forming the compound of Formula (XXb)comprises reacting the compound of Formula (XXV) with a suitableprotecting group removing agent.

Another embodiment relates to a process for preparation of a compound ofFormula (XXb) that further includes:

-   -   providing a compound of Formula (XXII):

-   -   and    -   forming the compound of Formula (XXV) from the compound of        Formula (XXII).

Another embodiment relates to a process for preparation of a compound ofFormula (XXb), wherein said forming the compound of Formula (XXV)comprises reacting the compound of Formula (XXII) with a compound ofFormula (XXVI):

-   -   wherein Z is halogen.

Yet another embodiment relates to a process for preparation of acompound of Formula (XXb) that further includes:

-   -   providing a compound of Formula (XXIV):

-   -   and    -   forming the compound of Formula (XXII) from the compound of        Formula (XXIV).

A further embodiment relates to a process for preparation of a compoundof Formula (XXb), wherein said forming the compound of Formula (XXII)comprises reacting the compound of Formula (XXIV) with a suitableprotecting group introducing agent, under conditions effective toproduce the compound of Formula (XXII).

EXAMPLES Materials for Examples 1-6

Di-tert-butyl dicarbonate, triethylamine, N,N-dimethylethylenediamine,barium chloride, magnesium sulfate, magnesium chloride hexahydrate,HEPES buffer, diethyl ether, ethyl alcohol, acetonitrile, anddichloromethane (DCM) were obtained from Sigma-Aldrich.2-Chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), N-methylmorpholine (NMM),1,3-propanesultone, and trifluoroacetic acid (TFA) were purchased fromthe Alfa Aesar. All the sodium alginates including VLVG (>60% G, 25 kDaMW), SLG20 (>60% G, 75-220 kDa MW), and SLG100 (>60% G, 200-300 kDa MW),were purchased from FMC BioPolymer Co. (Philadelphia, PA).Cyano-functionalized silica was purchased from SiliCycle.

Immuno-competent male C57BL/6 mice were obtained from Jackson Lab andSprague-Dawley rats were obtained from Charles River Laboratories. Allanimal procedures were approved by the Cornell Institutional Animal Careand Use Committee

Example 1—Synthesis of Zwitterionic Sulfobetaine-Based andCarboxybetaine-Based Alginate Conjugates

Di-tert-butyl dicarbonate (10.0 g, 45.8 mmol) and triethylamine (12.8mL, 91.6 mmol) were added dropwise over 0.5 hours to a solution ofN,N-dimethylethylenediamine (4.04 g, 45.8 mmol) in ethyl alcohol (150mL) at 0° C. The mixture was stirred for 1 hour at 0° C. and then for 18hours at room temperature. The white precipitate was filtered off andthe filtrate was evaporated to obtain residue. The residue was dissolvedin dichloromethane (150 mL), and the solution was washed successivelywith water. The organic layer was dried over anhydrous magnesium sulfateand evaporated to get N,N-dimethyl-2-((pivaloyloxy)amino)ethan-1-amine.

Example 2—Synthesis of Sulfobetaine-NH₂ Material

N,N-dimethyl-2-((pivaloyloxy)amino)ethan-1-amine (40.0 mmol),1,3-propanesultone (4.9 g, 40.0 mmol), and acetonitrile (150 mL) wereadded into a 250 mL round-bottom flask. The mixture was stirred undernitrogen atmosphere for 48 hours at 40° C. After reaction, the solventwas removed by rotary evaporator. The product was precipitated byanhydrous diethyl ether and washed with anhydrous diethyl ether to getwhite powder. Finally, 10.0 g of the obtained product was treated with amixture of 20 mL trifluoroacetic acid (TFA) and 20 mL dichloromethaneovernight at room temperature, concentrated with rotary evaporator,precipitated in anhydrous diethyl ether to get white power(sulfobetaine-NH₂ material). ¹H NMR (D₂O, 400 MHz): δ 3.70 (t, 2H), 3.54(m, 4H), 3.20 (s, 6H), 2.97 (t, 2H), 2.24 (m, 2H).

Example 3—Synthesis of Carboxybetaine-NH₂ Material

N,N-Dimethyl-2-((pivaloyloxy)amino)ethan-1-amine (40.0 mmol), tert-butylbromoacetate (40.0 mmol), and acetonitrile (150 mL) were added into a250 mL round-bottom flask. The mixture was stirred under nitrogenatmosphere for 48 hours at 40° C. After reaction, the solvent wasremoved by rotary evaporator. The product was precipitated by anhydrousdiethyl ether and washed with anhydrous diethyl ether to get whitepowder. Finally, 10.0 g of the obtained product was treated with amixture of 40 mL trifluoroacetic acid (TFA) and 40 mL dichloromethaneovernight at room temperature, concentrated with rotary evaporator,precipitated in anhydrous diethyl ether to get white power(carboxybetaine-NH₂ material). ¹H NMR (D₂O, 400 MHz): δ 4.31 (s, 2H),3.99 (m, 2H), 3.55 (m, 2H), 3.36 (s, 6H).

Example 4—Sulfobetaine-Based and Carboxybetaine-Based AlginateConjugation

VLVG alginate (0.5 g) was dissolved in the 40 ml of mixture solvent (30ml of DI water and 10 ml acetonitrile).2-Chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) (225 mg) andN-methylmorpholine (NMM) (280 μl) were added. Then 0.84 g ofsulfobetaine-NH₂ material was dissolved in 10 ml DI water and added intothe mixture. The reaction was stirred overnight at 55° C. The solventwas removed under reduced pressure and the solid product was redissolvedin DI water. The solution was filtered through a pad ofcyano-functionalized silica. It was then dialyzed against a 10,000 MWCOmembrane in DI water for three days. Finally, the water was removedunder reduced pressure to obtain sulfobetaine-modified alginate. Thecarboxybetaine-modified alginate was synthesized using the similarprocedure as that of sulfobetaine-modified alginate.

Example 5—Preparation of the Zwitterionic Sulfobetaine-Based AlginateMicrocapsules

To prepare alginate solution, 2% (w/v) alginate (VLVG, SLG20, or SLG100)was dissolved in 0.8% (w/v) NaCl solution. 2% (w/v) sulfobetaine-basedalginate conjugate was dissolved in 0.8% (w/v) NaCl solution. Themixture of 80% (by volume) sulfobetaine-based alginate conjugate and 20%(by volume) SLG100 were blended to obtain the sulfobetaine-alginate (orSA) solution.

All the buffers were sterilized before use and alginate solutions weresterilized by filtration through a 0.2 μm filter. Alginate hydrogelmicrocapsules were prepared via the method of electrospraying. Briefly,a high voltage power generator was connected to a blunt-tipped needle.This needle was attached to a 3 ml syringe which was clipped to asyringe pump that was oriented vertically. The syringe pump pumpedalginate solution out into a sterile, grounded dish containing a 20 mMbarium chloride solution. After the alginate microcapsules were formed,they were collected and then washed with prepared buffer (NaCl 15.43 g,KCl 0.70 g, MgCl₂·6H₂O 0.49 g, 50 ml of HEPES (1 M) buffer solution in 2L of DI water) 3 times. The alginate capsules were left overnight at 4°C. The capsules were finally washed 2 times in 0.8% saline and kept at4° C. before use.

Example 6—Implantation of the Alginate Microcapsules

The mice were anesthetized using 3% isoflurane in oxygen and maintainedat the same rate throughout the procedure. The abdomens of the mice wereshaved and alternately scrubbed with betadine and isopropyl alcohol tocreate a sterile field before being transferred to the surgical field. A˜0.5 mm incision was made along the midline of the abdomen and theperitoneum was exposed using blunt dissection. The peritoneum was thengrasped with forceps and a 0.5-1 mm incision was made along the lineaalba. A volume of ˜300 μl of microcapsules was then loaded into asterile pipette and implanted into the peritoneal cavity through theincision.

The phase contrast images of the microcapsules were taken using an EVOSAMF4300 imaging system.

Results and Discussion of the Examples 1-6

Among zwitterionic groups, sulfobetaine and carboxybetaine were chosenas examples. These zwitterionic materials have previously been shown toexhibit excellent antifouling properties (Zhang et al., “SuperlowFouling Sulfobetaine and Carboxybetaine Polymers on Glass Slides,”Langmuir 22(24):10072-10077 (2006), which is hereby incorporated byreference in its entirety). The sulfobetaine-NH₂ and carboxybetaine-NH₂monomers were synthesized according to Scheme 6. The structures of themonomers were confirmed by nuclear magnetic resonance (NMR), and thechemical shifts of the corresponding protons were marked in FIGS. 1A-B.

The sulfobetaine-NH₂ and carboxybetaine-NH₂ monomers were prepared usingN,N-dimethylethylenediamine and either (a) propanesultone or (b)tert-butyl bromoacetate.

To modify the alginate, low molecular weight (MW), VLVG alginate wasused as the starting material. 2-Chloro-4,6-dimethoxy-1,3,5-triazine(CDMT) and N-methylmorpholine (NMM) were used as coupling reagents toconjugate alginate with sulfobetaine-NH₂ via a triazine-based couplingreaction as shown in FIG. 2A. The sulfobetaine-based alginate conjugatewas characterized by ¹H NMR spectrum (FIG. 2B). The characteristic peakof sulfobetaine-NH₂ segment is as follows: the peak at 3.20 ppm isattributed to six protons in two methyl groups attached to thequaternary amine in the sulfobetaine pendant group. The result suggeststhat sulfobetaine-NH₂ was successfully conjugated to alginate. About30.5% modification of the starting alginate was confirmed by NMR dataanalysis. Carboxybetaine-based alginate was prepared as shown in FIG.3A. For the carboxybetaine-based alginate, characteristic peak of 3.25ppm in FIG. 3B was attributed to six protons in two methyl groupsattached to the quaternary amine in the carboxybetaine pendant group,and the modification degree is about 35.6%.

Since hydrogel microcapsules have been used extensively for cellencapsulation, culture, and transplantation, the microcapsules ofsulfobetaine-based alginate conjugate were fabricated by electrosprayingusing barium chloride as cross-linker, although other polyvalent ionssuch as Ca²⁺, Sr²⁺ could be used, which were previously demonstrated tohave long-term in vivo stability. The foreign body response to the SAmicrocapsules was evaluated in the intraperitoneal space of C57BL/6Jmice 14 days post implantation, with SLG20 alginate as control.

The whiteness on the microcapsule surfaces (FIG. 4 , top row of images)indicated the fibrotic deposition, and therefore the controlmicrocapsule (SLG 20) induced substantial fibrosis. In contrast, therewas almost no fibrotic deposition on the sulfobetaine modified VLVG(SB-VLVG) microcapsules (FIG. 4 , second row of images from top).Moreover, sulfobetaine modified SLG 20 (SB-SLG20) and sulfobetainemodified SLG 100 (SB-SLG 100) microcapsules also exhibited almost nofibrosis (FIG. 4 , third and fourth rows of images from the top,respectively). These results suggested that the SB modified alginatemicrocapsules, regardless of alginate types, mitigated the foreign bodyresponse effectively and reproducibly.

To investigate the effect of SB modified alginate on the foreign bodyreactions in the long-term, SB-SLG 20 microcapsules were implanted intothe intraperitoneal space of immunocompetent C57BL/6J mice and harvestedafter 100 days. Dark field phase contrast microscopy of retrieved SLG 20microcapsules showed extensive fibrotic deposition (FIG. 5 , top row ofimages), whereas SB-SLG20 microspheres showed much lower level offibrotic deposition (FIG. 5 , bottom row of images). Moreover, some ofretrieved SLG 20 microcapsule even aggregated together, a sign of severefibrosis (FIG. 5 , top row, third image from left).

To further evaluate performance of SB modified alginate, SB-SLG20microcapsules were examined after 180 days implantation in C57BL/6J. Asshown in FIG. 6 , SB-SLG 20 microcapsules after retrieval were largelyclean of fibrotic deposition (FIG. 6 , top two rows of images) whilethere was substantial fibrosis observed on the conventional SLG 20microcapsules surfaces (FIG. 6 , bottom two rows of images). FIG. 7shows numerous retrieved microcapsules in each dish. The whiteness onthe microcapsule surfaces indicated the fibrotic deposition. Clearly,the whiteness mostly shown on the SLG 20 microcapsules indicated severefibrosis (FIG. 7 , top two rows); the absence of this whiteness for theSB-SLG 20 microcapsules indicated almost no fibrosis (FIG. 7 , bottomtwo rows). This result suggested that SB modified alginate substantiallyreduced foreign body reactions in the intraperitoneal space of C57BL/6mice for a significantly longer period of 180 days.

Another type of sulfobetaine modified alginate: ethylene-glycol SBalginate was also designed and developed. The foreign body response tothe ethylene-glycol SB-SLG20 microcapsules was evaluated in theintraperitoneal space of C57BL/6J mice 30 days post implantation. Afterretrieval, ethylene-glycol SB-SLG20 microcapsules (FIG. 8 ) were clean,and showed almost no fibrosis.

Taken together, zwitterionic modified alginates were capable ofmitigating the foreign body response effectively and reproducibly. Thismay be attributed to the zwitterionic sulfobetaine group that reducesbiofouling and improves the biocompatibility of alginate. Thiszwitterionic sulfobetaine-based alginate conjugate will be applicable toislet encapsulation to support long-term diabetes correction for type-1diabetes (TD1).

After confirming that zwitterionically modified alginates such asSB-SLG20 resists fibrosis in C57BL/6J mice, its therapeutic potential asa cell encapsulation medium for the treatment of diabetes was explored.Encapsulated rat islets were transplanted into the peritoneal cavity ofstreptozotocin (STZ)-induced C57BL/6 diabetic mice and evaluated for 90days for their ability to restore normoglycemia. Rat islets wereencapsulated with 1000 μm SB-SLG20 microcapsules as barrier to shieldforeign body response, or with SLG 20 microcapsules as control (FIG.9A). The blood glucose (BG) level of the mice decreased to the normalglycemic range (BG<200 mg/dL) a few days after the transplantation (FIG.9B). However, mice transplanted with rat islets encapsulated in SLG 20microcapsules showed a shorter duration of glycemic control and wereunable to sustain normoglycemia beyond 45 days, while the micetransplanted with rat islets encapsulated in SB-SLG 20 microcapsulesremained cured for 3 months before the microcapsules were retrieved.

The whiteness was mostly shown on the retrieved SLG 20 microcapsules(FIG. 9C), suggesting severe fibrosis. It was also confirmed using H&Estained histological analysis (FIG. 9D). The result of H&E staining oftissue sections showed that SLG 20 produced microcapsules with highfibrotic deposition around the implant. On the contrary, retrievedSB-SLG 20 microcapsules encapsulating rat islets (FIG. 9E-F) showedalmost no fibrous deposition. There were also numerous rat isletsobserved in the microcapsules after retrieval. Importantly, these isletswere still functional after 3 months implantation, as indicated by thepositive insulin staining (FIG. 9G) on the islets in retrieved SB-SLG 20microcapsules. Taken together, encapsulated rat islets were able toachieve long-term glycemic correction (90 days) in STZ-treated C57BL/6Jmice for T1D, using the zwitterionically modified alginate capable ofmitigating the FBR effectively.

Super-biocompatible, zwitterionically modified alginates were designedand synthesized. Sulfobetaine-NH2, ethylene glycol sulfobetaine-NH2, andcarboxybetaine-NH2 were successfully synthesized and subsequentlyconjugated to alginate. The sulfobetaine-modified alginate were testedin C57BL/6 mice and mitigated the foreign body response effectivelycompared to the unmodified control alginate. The therapeutic potentialof the SB modified alginate was demonstrated through a type 1 diabeticmouse model using rat islets. SB-SLG 20 encapsulated islet cells wereproven to be capable of providing long-term glycemic correction inimmune-competent diabetic C57BL/6J mice. It was determined that thiszwitterionically modified alginate may contribute to the translation ofcell encapsulation for T1D and potentially other diseases.

Materials for Examples 7-16

Propargylamine, acryloyl chloride, sodium azide, sodium ascorbate,copper sulfate pentahydrate, iodomethane, trifluoroacetic acid (TFA),2-chloro-N,N-dimethylethylamine hydrochloride, 1,3-propanesultone,2-hydroxyethyl methacrylate (HEMA), 2-hydroxy-2-methylpropiophenone,phosphate buffered saline (PBS), dichloromethane (DCM), dimethylsulfoxide (DMSO), acetonitrile, hexane, ethyl acetate, diethyl ether,and ethyl alcohol were purchased from Sigma-Aldrich. Tert-butylbromoacetate and ion exchange resin (Amberlyst A-26, OH form) wereobtained from Alfa Aesar. Procedures for the synthesis of qTR-CB, TR-CB,and TR-SB monomers are described below. Carboxybetaine methacrylate (CB)(Yang et al., “Pursuing “Zero” Protein Adsorption ofPoly(carboxybetaine) from Undiluted Blood Serum and Plasma,” Langmuir25(19):11911-11916 (2009), which is hereby incorporated by reference inits entirety) and carboxybetaine diacrylamide (CBAAX) (Zhang et al.,“Zwitterionic gel Encapsulation Promotes Protein Stability, EnhancesPharmacokinetics, and Reduces Immunogenicity,” Proceedings of theNational Academy of Sciences 112(39):12046-12051 (2015), which is herebyincorporated by reference in its entirety) were synthesized usingmethods reported previously.

Example 7—Hydrogel Preparation

The TR-ZW hydrogels were prepared via radical polymerization initiatedby UV irradiation. The hydrogel solution consisted of 1 mL DI water, 600mg monomer, 4% CBAAX cross linker (molar percent of monomer), and 3.5 mg2-hydroxy-2-methylpropiophenone photo-initiator. The resulting solutionwas cast between a pair of glass slides, separated with a 2-mm-thicknesspoly(tetrafluoroethylene) (PTFE) spacer, and polymerized under UV (365nm) for 45 min. PHEMA and PCB hydrogels were prepared using a similarprocedure. After preparation, all hydrogel samples were equilibrated insterile PBS buffer and the PBS buffer solution was changed at leastthree times a day for five days. For implantation, the hydrogels werepunched into disks with a diameter of 6 mm, and stored in sterile PBS at4° C. before use.

Example 8—Protein Adsorption Assay

P(qTR-CB) polymer brushes were grafted onto gold-coated surfaceplasmonic resonance (SPR) sensor chips following the procedure reportedpreviously (Zhang et al., “Zwitterionic Hydrogels: an in vivoImplantation Study,” Journal of Biomaterials Science, Polymer Edition20(13):1845-1859 (2009), which is hereby incorporated by reference inits entirety). The protein adsorption on the P(qTR-CB)-grafted goldsurfaces was evaluated using a four-channel SPR sensor. Firstly, PBSbuffer was flowed into the channels for 10 min to build the baseline.Secondly, a 1 mg/mL fibrinogen solution or 100% human blood plasma wasrun through the channels for 10 min followed by a PBS buffer wash toremove unbound protein molecules. The amount of adsorbed protein wasfinally quantified by the change of wavelength shift between thepre-adsorptive and post-adsorptive baselines. A 1 nm SPR wavelengthshift at 750 nm corresponded to a protein surface coverage of 15 ng/cm²(Liu et al., “Amino Acid-Based Zwitterionic Poly(Serine Methacrylate) asan Antifouling Material,” Biomacromolecules 14(1):226-231 (2012), whichis hereby incorporated by reference in its entirety). The proteinadsorption on the P(TR-CB) and P(TR-SB) surfaces was evaluated using thesame procedure.

Example 9—Cell Attachment Assay

NIH/3T3 cells were cultured in a humidified incubator with 5% CO₂ at 37°C. The culture medium was composed of Dulbecco's modified Eagle medium(DMEM), 10% fetal bovine serum (FBS), and 2% penicillin streptomycin.The hydrogel disks with a diameter of 6 mm were individually placed intoa 12-well plate and washed with sterile PBS buffer three times. Cellsuspension (2 mL) (concentration: 105 cells/mL) was then transferredinto each well and incubated with these hydrogels for 3 days at 37° C.After incubation, the hydrogels were transferred to a new 12-well platecontaining sterile PBS in each well. The LIVE/DEAD assay solution wasadded into each well and incubated for 30 min. These hydrogels werefinally imaged by using an EVOS AMF4300 imaging system.

Example 10—Tensile and Compression Tests

Tensile tests of hydrogel samples were performed on a TA instrument DMAQ800 Dynamic Mechanical Thermal Analysis (DMTA). All equilibratedhydrogel samples were cut in rectangular shape with 25 mm length, 6 mmwidth, and 2-3 mm thickness. Hydrogel samples were stretched untilfailure at a rate of 5 mm min⁻¹. The compression tests andloading-unloading tests of hydrogel samples were done on Instron 5965with a 100 N load cell. For compression tests, each hydrogel disk with adiameter of 6 mm (about 2-3 mm thickness when equilibrated in PBSbuffer) was compressed until failure at a rate of 1 mm min⁻¹. The shaperecovery property of the hydrogels was evaluated by ten consecutiveloading and unloading cycles at a constant rate of 1 mm min⁻¹ in thestrain range of 0-65%. All samples were measured at the roomtemperature.

Example 11—Cytokine Secretion

Bone marrow derived macrophages (BMDMs) were seeded on the tissueculture plates or various hydrogel surfaces at a cell density of 10⁶cells/cm² and stimulated with different combinations: 0.3 ng/mLlipopolysaccharide (LPS) (Sigma-Aldrich), 1.0 ng/mL IFNγ (R&D systems,Minneapolis, MN), 20 ng/mL IL-4 (Invitrogen), and 20 ng/mL IL-13(Invitrogen). After stimulation for 36 hours, supernatants werecollected and analyzed for TNF-α and IL-10 secretion by ELISA(enzyme-linked immunosorbent assay) following the manufacturer'sinstructions (BioLegend, San Diego, CA).

Example 12—Hydrogel Implantation and Histological Analysis

All animal protocols were approved by the Cornell Institutional AnimalCare and Use Committee. Eight-week-old, immune-competent male C57BL/6mice were obtained from Jackson Laboratory. The equilibrated hydrogeldisks were implanted subcutaneously in mice for 1, 2, and 3 months,respectively. For each mouse, hydrogel disks made from differentmonomers were implanted on the back of the mouse and the sites ofvarious hydrogel samples were alternated in order to eliminate theeffect of implantation positions. At the end of each experiment, micewere euthanized by CO₂ asphyxiation. The hydrogel disks together withsurrounding tissues were dissected and fixed in 10% neutrally bufferedformalin. After embedded in paraffin wax, the samples were sectioned andstained with Masson's trichrome by Cornell Histology Core Facility. Thestained histology slides were scanned using an Aperio CS2 ScanScope(Leica Biosystems, Nusslock GmbH). The blue-pixel density was measuredusing an Image J software. The collagen density was quantified as apercentage of average maximum blue-pixel density as determined from allanalyzed sections. For each sample, three random fields were analyzedfor each fixed distance (e.g. 0 to 10 μm; 10 to 20 μm; etc.) within 60μm from the tissue-hydrogel interface. The sample size was n=5.

In order to assess the blood vessel formation upon the hydrogels,paraffin-embedded sections were stained using primary Goat anti-mouseCD31 (R&D Systems, dilution 1:200), which is an endothelial cellbiomarker. The Alexa Fluor 488 donkey anti-Goat as secondary antibody(Life Technologies, dilution 1:500) was used in this work. Two stainedsections at different positions of each hydrogel disks were used andfive different fields were randomly examined in each section. Thedensity of blood vessels were quantified by counting the number of thevascular features normalized to the capsule area. The sample size wasn=5.

Example 13—Statistical Analysis

All the data are presented as the mean value±standard deviation. Thedifference was considered statistically significant when the p value isless than 0.05. A Student's t-test was used for all statisticalanalysis.

Example 14—Synthesis of qTR-CB Monomer

N-Propargylacrylamide (10a)

Propargylamine (3.3 g, 60 mmol) was dissolved in 150 mL DCM at 0° C.Aqueous NaOH (1.5 M, 100 mL) was then added into the solution. Acryloylchloride (14.9 g, 165 mmol) was added dropwise into the denserdichloromethane layer over 30 minutes resulting in a yellow orangesolution. The mixture was stirred for 1 hour at 0° C. and then for 18hours at room temperature. The resulting solution was washedsuccessively with water. The organic layer was dried over anhydroussodium sulfate and evaporated to get yellow oil. The productN-propargylacrylamide (4.2 g, 64%) was obtained as light yellow solid bysilica gel column chromatography (eluent: ethyl acetate/hexane, 1:1,v/v). ¹H NMR (CDCl₃, 400 MHz): δ 6.32 (dd, 1H), 6.13 (dd, 1H), 5.68 (dd,1H), 4.12 (m, 2H), 2.23 (m, 1H) (FIG. 10 ). ¹³C NMR (CDCl₃, 400 MHz):165.4, 130.1, 127.2, 79.3, 71.6, 29.2.

Tert-butyl 2-azidoacetate (15a)

Tert-butyl bromoacetate (15.6 g, 80 mmol) was dissolved in 100 mL DMSOat room temperature. Sodium azine (NaN₃) (6.5 g, 100 mmol) was addedslowly into the solution and stirred for overnight at 70° C. Water (150mL) was added to quench the reaction and the water layer was extractedwith 3×200 mL anhydrous diethyl ether. The combined organic phase waswashed successively with brine and dried over anhydrous sodium sulfate.The organic solvent was removed under reduced pressure to obtain theproduct of tert-butyl 2-azidoacetate (14.2 g, 90%). ¹H NMR (CDCl₃, 400MHz): δ 3.74 (s, 2H), 1.49 (s, 9H) (FIG. 11 ). ¹³C NMR (CDCl₃, 400 MHz):167.3, 82.9, 50.8, 27.9.

Tert-butyl 2-(4-(acrylamidomethyl)-1H-1,2,3-triazol-1-yl)acetate (16a)

The mixture of product 10a (4.2 g, 38.4 mmol), product 15a (6.1 g, 38.4mmol), sodium ascorbate (0.76 g, 3.8 mmol), CuSO4·5H2O (0.96 g, 3.8mmol), and 100 mL DMSO were added into a 250 mL round-bottom flask. Themixture was stirred under nitrogen atmosphere for 48 hours at 60° C. 150mL water was added to quench the reaction. Then the resulting solutionwas extracted with ethyl acetate three times. The combined organic layerwas dried over anhydrous sodium sulfate and the solvent was removedunder reduced pressure to get the crude product. The product 16a (8.0 g,78%) was further purified by silica gel column chromatography (eluent:ethyl acetate). ¹H NMR (CDCl₃, 400 MHz): δ 7.68 (s, 1H), 6.27 (dd, 1H),6.13 (dd, 1H), 5.63 (dd, 1H), 5.01 (s, 2H), 4.57 (m, 2H), 1.45 (s, 9H)(FIG. 12 ). ¹³C NMR (CDCl₃, 400 MHz): 165.5, 165.1, 144.7, 130.4, 126.4,123.9, 83.6, 51.3, 34.5, 27.7.

4-(Acrylamidomethyl)-1-(2-(tert-butoxy)-2-oxoethyl)-3-methyl-1H-1,2,3-triazol-3-ium(17a)

Product 16a (8.0 g, 30 mmol), iodomethane (25.6 g, 180 mmol), andacetonitrile (150 mL) were added into a 250 mL round-bottom flask. Themixture was stirred under nitrogen atmosphere for 48 hours at 60° C.After reaction, the solvent was removed by rotary evaporator. Theresulting product was precipitated by anhydrous diethyl ether and washedwith anhydrous diethyl ether to get tan powder of product 17a (7.5 g,61%). ¹H NMR (D₂O, 400 MHz): δ 8.58 (s, 1H), 6.26 (dd, 2H), 5.83 (dd,1H), 5.48 (s, 2H), 4.74 (s, 2H), 4.34 (s, 3H), 1.47 (s, 9H) (FIG. 13 ).¹³C NMR (D₂O, 400 MHz): 168.7, 164.8, 141.0, 129.0, 128.9, 128.7, 86.3,54.2, 38.0, 32.2, 27.0.

Quaternized Triazole Carboxybetaine Acrylate (qTR-CB)

The obtained product 17a (7.5 g) was treated with a mixture of 15 mLtrifluoroacetic acid (TFA) and 15 mL DCM overnight at room temperature,concentrated by rotary evaporator, precipitated in anhydrous diethylether, and redissolved in methanol. Ion-exchange resin (Amberlyst A26,OH-form) was added into it for complete neutralization. The residue wasdissolved in water and lyophilized by freeze dryer to give productqTR-CB. (2.2 g, 54%)¹H NMR (D₂O, 400 MHz): δ 8.49 (s, 1H), 6.28 (dd,2H), 5.82 (dd, 1H), 5.22 (s, 2H), 4.74 (s, 2H), 4.30 (s, 3H) (FIG. 14 ).¹³C NMR (D₂O, 400 MHz): 170.0, 168.7, 140.6, 130.0, 128.9, 128.6, 55.6,37.6, 32.1.

Example 15—Synthesis of TR-CB Monomer

2-Azido-1-ethyl-dimethylamine (8a)

NaN₃ (13.7 g, 210 mmol) was added into a solution of2-chloro-N,N-dimethylethylamine hydrochloride (10.0 g, 70 mmol) in 100mL water and the reaction mixture was heated to 70° C. for overnight.The solution was basified with 4 M NaOH solution and extracted withanhydrous diethyl ether three times. The resulting solution was driedover MgSO4 and concentrated to give volatile colorless oil. (5.9 g,74%)¹H NMR (CDCl₃, 400 MHz): δ 3.31 (t, 2H), 2.45 (t, 2H), 2.21 (s, 6H,N(CH₃)₂).

N-(2-Azidoethyl)-2-(tert-butoxy)-N,N-dimethyl-2-oxoethan-1-aminium (9a)

Product 8a (5.9 g, 52 mmol), tert-butyl bromoacetate (12.7 g, 65 mmol),and acetonitrile (100 mL) were added into a 250 mL round-bottom flask.The mixture was stirred under nitrogen atmosphere for 24 hours at 60° C.After reaction, the solvent was removed by rotary evaporator. Theproduct 9a was precipitated by anhydrous diethyl ether and washed withanhydrous diethyl ether to get white powder (12.6, 79%). ¹H NMR (D₂O,400 MHz): δ 4.29 (s, 2H), 3.98 (t, 2H), 3.83 (t, 2H), 3.34 (s, 6H), 1.53(s, 9H) (FIG. 15 ). ¹³C NMR (D₂O, 400 MHz): δ 164.0, 86.3, 62.6, 62.1,52.9, 44.7, 27.2.

N-(2-(4-(Acrylamidomethyl)-1H-1,2,3-triazol-1-yl)ethyl)-2-(tert-butoxy)-N,N-dimethyl-2-oxoethan-1-aminium(11a)

The mixture of product 10a (4.9 g, 45.1 mmol), product 9a (12.6 g, 41.0mmol), sodium ascorbate (0.8 g, 4 mmol), CuSO4·5H2O (1.0 g, 4 mmol), and100 mL methanol were added into a 250 mL round-bottom flask. The mixturewas stirred under nitrogen atmosphere for 48 hours at 60° C. After thereaction, the solvent was removed by rotary evaporator and the crudeproduct was precipitated by anhydrous diethyl ether. The product 1a(11.7 g, 68%) was further purified by silica gel column chromatography(eluent: methanol). ¹H NMR (D₂O, 400 MHz): δ 8.11 (s, 1H), 6.24 (dd,2H), 5.75 (dd, 1H), 5.07 (t, 2H), 4.58 (s, 2H), 4.28 (m, 4H), 3.34 (s,6H), 1.44 (s, 9H) (FIG. 16 ). ¹³C NMR (D₂O, 400 MHz): 168.1, 163.3,145.4, 129.3, 127.4, 124.7, 86.6, 62.3, 62.1, 52.7, 48.8, 44.0, 34.3,27.1.

Triazole Carboxybetaine Acrylate (TR-CB)

The obtained product 11a (8.0 g) was treated with a mixture of 16 mL TFAand 16 mL DCM overnight at room temperature, concentrated with rotaryevaporator, precipitated in anhydrous diethyl ether, and dissolved inmethanol. Ion-exchange resin (Amberlyst A26, OH-form) was then addedinto it for complete neutralization. The resulting solution was addedinto neutral alumina column to remove residual copper ion. The productTR-CB (3.1 g, 57%) was collected after removing methanol solvent. ¹H NMR(D₂O, 400 MHz): δ 8.02 (s, 1H), 6.22 (dd, 2H), 5.75 (dd, 1H), 4.98 (t,2H), 4.51 (s, 2H), 4.24 (t, 2H), 3.89 (s, 2H), 3.24 (s, 6H) (FIG. 17 ).¹³C NMR (D₂O, 400 MHz): 168.3, 168.1, 145.1, 129.5, 127.7, 124.2, 63.9,61.3, 51.9, 44.0, 34.3.

Example 16—Synthesis of TR-SB Monomer

N-((1-(2-(Dimethylamino)ethyl)-1H-1,2,3-triazol-4-yl)methyl)acrylamide(12a)

The mixture of product 10a (4.9 g, 45.1 mmol), product 8a (4.7 g, 40mmol), sodium ascorbate (0.8 g, 4 mmol), CuSO4·5H2O (1.0 g, 4 mmol), and100 mL methanol were added into a 250 mL round-bottom flask. The mixturewas stirred under nitrogen atmosphere for 48 hours at 60° C. After thereaction, the solvent was removed by rotary evaporator. The product 12a(8.0 g, 78%) was further purified by silica gel column chromatography(eluent: ethyl acetate/methanol, 1:1, v/v). ¹H NMR (CDCl₃, 400 MHz): δ7.68 (s, 1H), 7.24 (s, 1H), 6.19 (dd, 2H), 5.60 (dd, 1H), 4.54 (m, 2H),4.38 (m, 2H), 2.71 (m, 2H), 2.23 (s, 6H) (FIG. 18 ). ¹³C NMR (CDCl₃, 400MHz): 165.6, 144.3, 130.6, 126.7, 122.9, 58.7, 48.2, 45.4, 34.8.

Triazole Sulfobetaine Acrylate (TR-SB)

Product 12a (4.5 g, 20 mmol) in 50 mL anhydrous acetone was stirred atroom temperature. 1,3-Propanesultone (20 mmol, 2.4 g) was added dropwiseinto the solution. The reaction mixture was heated to 40° C. undernitrogen atmosphere for 6 hours. The precipitate was collected andwashed with anhydrous acetone to get white powder (TR-SB) (2.8 g, 41%).¹H NMR (D₂O, 400 MHz): δ 8.06 (s, 1H), 6.26 (dd, 2H), 5.79 (dd, 1H),5.02 (m, 2H), 4.55 (s, 2H), 4.01 (m, 2H), 3.53 (m, 2H), 3.17 (s, 6H),2.90 (m, 2H), 2.18 (m, 2H) (FIG. 19 ). ¹³C NMR (D₂O, 400 MHz): 168.4,145.2, 129.5, 127.7, 124.2, 63.0, 61.6, 51.1, 46.9, 43.7, 34.3, 18.1.

Results and Discussion of Examples 7-16

A new zwitterionic monomer qTR-CB was designed (FIG. 20A). This monomerincludes a triazole moiety that plays a critical role in theanti-fibrotic properties of modified alginates and formsenergy-dissipating π-π stacking. As shown in FIG. 20B, the synthesis ofqTR-CB involved several steps. First, N-propargylacrylamide with dualreactive alkyne and vinyl groups was developed. Next, the alkyne groupwas then transformed into triazole group through Azide-Alkyne HuisgenCycloaddition chemistry followed by a subsequent quaternization. Finallythe qTR-CB monomer was obtained after removal of the protecting group ofthe carboxylic acid. The chemical structure of the qTR-CB monomer wasconfirmed by ¹H NMR (FIG. 17 ) and ¹³C NMR.

Non-specific protein adsorption on the implant surface is considered theinitial, critical step in the foreign body response. To determinewhether the qTR-CB was anti-biofouling and resistant to non-specificprotein adsorption, a gold surface was grafted with P(qTR-CB) using asurface-initiated photoiniferter-mediated polymerization. Theprotein-resistance of P(qTR-CB) was evaluated via surface plasmonresonance (SPR) using a single protein solution and an undiluted humanplasma. FIG. 20C shows the typical SPR sensorgrams of protein adsorptionon the P(qTR-CB)-grafted and bare gold surfaces. From a 1 mg/mLfibrinogen (Fg) solution, the bare gold and P(qTR-CB)-grafted surfaceshad adsorptions of 337.5±36.1 and 0.6±0.3 ng/cm², respectively. Fromundiluted human plasma, the protein adsorptions were 211.6±10.3 and3.1±1.8 ng/cm² respectively for these two surfaces. Clearly, theP(qTR-CB)-grafted surface was highly resistant to non-specific proteinadsorption, as compared to the bare gold surface. It should be notedthat protein adsorption values on the P(qTR-CB)-grafted surfaces werewell below the criteria for ultralow-fouling materials (less than 5ng/cm² adsorbed fibrinogen). These data suggest that incorporation oftriazole group did not change the zwitterionic or anti-foulingproperties. The P(qTR-CB) hydrogel (FIG. 20D) was then prepared bycrosslinking the qTR-CB monomer with a bifunctional zwitterioniccarboxybetaine diacrylamide cross-linker (CBAAX) via a photo-initiatedpolymerization.

The P(qTR-CB) hydrogel was designed to address the mechanical propertychallenge faced by current zwitterionic hydrogels which are known to berelatively brittle or weak (Chin et al., “Additive Manufacturing ofHydrogel-Based Materials for Next-Generation Implantable MedicalDevices,” Science Robotics 2(2):eaah6451 (2017); Lynn et al.,“Characterization of the in Vitro Macrophage Response and in Vivo HostResponse to Poly (Ethylene Glycol)-Based Hydrogels,” Journal ofBiomedical Materials Research Part A 93(3):941-953 (2010), which arehereby incorporated by reference in their entirety). Robust mechanicalproperties are highly desirable for handling, implantation and anyfuture clinical applications. The reversible π-π stacking between thetriazole rings within the P(qTR-CB) hydrogel (FIG. 21A) dissipatesenergy under load and therefore make the hydrogel more resilient. Todetermine whether the incorporation of the triazole rings indeedimproved the mechanical property, the P(qTR-CB) and conventional PCBhydrogels were compared in several mechanical tests. First, theirfold-resistance property was qualitatively examined. As shown in FIG.21B, the P(qTR-CB) hydrogel could be completely folded close to 180degree without fracturing or any damage, and was even amenable torepeated folding. In contrast, the conventional PCB hydrogel fracturedeven with a small-angle folding. More quantitative tensile andcompression tests were then performed. For the tensile test, theP(qTR-CB) hydrogel had a breaking strain close to 71% while the PCBhydrogel could only be stretched 11% (FIG. 21C). That represented a6.5-fold increase in the breaking strain. For the compressive test (FIG.21D), the P(qTR-CB) hydrogel sustained a 80% compression, while the PCBhydrogel could only be compressed 48%, which was in agreement withprevious work (Merino et al., “Nanocomposite Hydrogels: 3DPolymer-Nanoparticle Synergies for On-Demand Drug Delivery,” ACS nano9(5):4686-4697 (2015), which is hereby incorporated by reference in itsentirety). To further demonstrate the resilience of the P(qTR-CB)hydrogel, a compressive loading-unloading test was performed. As shownin FIG. 21E, the P(qTR-CB) hydrogel tolerated 65% compression for atleast 10 cycles without any crack and maintained its original shape. Thehysteresis loop observed in each cycle seemed to suggest that there wasan energy dissipation mechanism probably due to the π-π stacking betweenthe triazole groups, confirming the hypothesis.

Next, cell attachment and macrophage activation on the P(qTR-CB)hydrogel was investigated in vitro. Hydrogels that resist cellattachment are desirable for many biomedical applications. Cellattachment on the P(qTR-CB) hydrogel was studied by culturing NIH/3T3fibroblasts on its surface for three days. For comparison, PHEMA and PCBhydrogels as well as tissue culture polystyrene (TCPS) were used ascontrols. FIG. 22A showed that cells quickly attached, proliferated, andformed a confluent layer on the TCPS surfaces while there were almost nocells observed on the P(qTR-CB), PHEMA, and PCB hydrogel surfaces,suggesting that P(qTR-CB) hydrogel behaved similarly to PHEMA and PCB.The macrophage activation was then explored. Macrophages as a keycomponent of the FBR regulate pro-inflammatory or pro-healing processes.Pro-inflammatory macrophages secrete inflammatory cytokines such astumor necrosis factor-α (TNF-α) that triggers further recruitment andactivation of inflammatory cells, while pro-healing macrophages produceanti-inflammatory cytokines such as interleukin (IL-10) that facilitatesangiogenesis and tissue repair. Understanding how a biomaterialregulates macrophage phenotype is of importance to its biomedicalapplications. As shown in FIG. 22B, the levels of TNF-α and IL-10secretion were almost undetectable for all the hydrogels withoutstimulation. With the stimulation of lipopolysaccharide/Interferon gamma(LPS/IFNγ) which was known to induce a pro-inflammatory macrophagephenotype, the cells on the PCB and P(qTR-CB) hydrogels secreted lowerlevels of TNF-α when compared to those cultured on the PHEMA hydrogel orthe TCPS. With the stimulation of LPS/IL-4/IL-13 which was known topromote a pro-healing macrophage phenotype, the cells on PCB andP(qTR-CB) hydrogels had an enhanced IL-10 secretion when compared tothose on the PHEMA hydrogel. Taken together, these results showed thatP(qTR-CB) hydrogels inhibited inflammatory activation and promotedpro-healing macrophage phenotype.

Encouraged by the results obtained from the P(qTR-CB), two morecrosslinkable, triazole-containing zwitterionic monomers, TR-CB andTR-SB, were designed (FIG. 23A and FIG. 25 ). Hydrogels from thesemonomers were made and tested for FBR in mice. The synthetic routes forthe TR-CB or TR-SB monomers are shown in FIG. 23A, and their chemicalstructures were confirmed by ¹H NMR (FIGS. 17 and 19 ) and ¹³C NMR.Compared to the qTR-CB in which the triazole moiety was quaternized, theTR-CB and TR-SB monomers have an original, un-modified triazole group.The mechanical properties of the P(TR-CB) and P(TR-SB) hydrogels wereevaluated. As shown in FIG. 23B, the P(TR-CB) and P(TR-SB) hydrogelscould endure repeated folding, similar to that of P(qTR-CB) hydrogels.Both hydrogels were highly resilient (FIG. 23C-23E). Especially for theP(TR-SB) hydrogel (FIG. 23C-23D), the tensile strain was as high as218%, while the maximum tensile strain for zwitterionic hydrogelsreported to date is only 65% (Lynn et al., “Characterization of the inVitro Macrophage Response and in Vivo Host Response to Poly (EthyleneGlycol)-Based Hydrogels,” Journal of Biomedical Materials Research PartA 93(3):941-953 (2010), which is hereby incorporated by reference in itsentirety). This is a drastic improvement in the field of zwitterionichydrogels. When compared to PCB hydrogel, the breaking strains ofP(TR-CB) and P(TR-SB) hydrogels were 9-fold and 20-fold higher,respectively. It should be also noted that P(TR-CB) and P(TR-SB)hydrogels were more elastic than the P(qTR-CB). This may be attributedto the position of the positive charge on the qTR-CB triazole ring. Theelectrostatic repulsion between the charges may attenuate the π-πstacking interaction. For compressive tests (FIG. 23E), both theP(TR-CB) and P(TR-SB) hydrogels had high compressive strains (79% and83%, respectively). These TR-ZW hydrogels represent a first class ofzwitterionic hydrogel with robust mechanical properties.

To investigate whether these new TR-ZW hydrogels (P(qTR-CB), P(TR-CB),and P(TR-SB)) had FBR-resistant properties, they were subcutaneouslyimplanted in immunocompetent C57BL/6 mice. To date, the FBR is still amajor concern for the performance and longevity of implanted materialsand devices. There is a critical need for development of novel materialsthat mitigate FBR and are mechanically robust. In the presentapplication, the FBR to the implants at selected time points postimplantation (1, 2, and 3 months) were evaluated. A commonly used PHEMAhydrogel was chosen as control. At each time point, the hydrogel sampleswere retrieved and examined the FBR including the fibrosis around theimplants using Masson's trichrome staining as well as the blood vesselformation using CD31 staining. At 1 month, it was observed that allTR-ZW hydrogels had loose collagen layers around them as indicated bythe light blue color (FIG. 24A), while the PHEMA hydrogels had a muchdenser collagen deposition. The P(TR-SB) hydrogel had a particularly lowdensity collagen deposition. The loose collagen deposition has beenthought to affect less the mass transfer between the body and implantand therefore is desirable in many applications. Longer-termimplantation experiments (i.e. 2 and 3 months) revealed similar results.The collagen density at the interface between the TR-ZW hydrogels andtissues was significantly lower when compared to the case of PHEMAcontrol (FIG. 24B).

When comparing the TR-ZW hydrogels with the previously reported PCBhydrogels (Jiang et al., “Click Hydrogels, Microgels and Nanogels:Emerging Platforms for Drug Delivery and Tissue Engineering,”Biomaterials 35(18):4969-4985 (2014); Lee et al., “Light-Triggered inVivo Activation of Adhesive Peptides Regulates Cell Adhesion,Inflammation and Vascularization of Biomaterials,” Nature Materials14(3):352-360 (2015), which are hereby incorporated by reference intheir entirety), it was found that the density of collagen depositionand the number of blood vessels for the TR-ZW hydrogels were comparableto or even better than those for the PCB (Jiang et al., “ClickHydrogels, Microgels and Nanogels: Emerging Platforms for Drug Deliveryand Tissue Engineering,” Biomaterials 35(18):4969-4985 (2014), which ishereby incorporated by reference in its entirety) (FIGS. 26A-26B). It isgenerally held that the antifouling property or biocompatibility iscompromised if hydrophobic moiety is incorporated into zwitterionicmaterials. The triazole group as a hydrophobic moiety indeed affectedits antifouling property. For example, the amount of plasma adsorptionfor P(qTR-CB), P(TR-CB), and P(TR-SB) surfaces was 3.1±1.8, 6.4±2.5, and10.9±3.2 ng/cm2, respectively (Table 1) while PCB surface was reportedto only adsorb <0.3 ng/cm2 protein from plasma. However, the in vivobiocompatibility or the FBR-resistant property of the TR-ZW hydrogelswas not affected. The triazole group plays a role here in mitigating thefibrotic response in addition to the zwitterionic moiety. Chemicallymodified alginates containing triazole groups were reported to becapable of mitigating the fibrotic response effectively (Desai et al.,“Versatile Click Alginate Hydrogels Crosslinked via Tetrazine-NorborneneChemistry,” Biomaterials 50:30-37 (2015), which is hereby incorporatedby reference in its entirety). The FBR-resistant property of the TR-ZWhydrogels was also consistent with the macrophage activation data (FIG.22B). Thus, a new class of hydrogels was developed that exhibitedsimilar FBR-resistant properties but were much more mechanically robustthan the zwitterionic hydrogels developed to date.

TABLE 1 Protein Adsorption (Undiluted human plasma) Measured by SPRSurface Bare gold P(qTR-CB) P(TR-CB) P(TR-SB) Surface mass 211.6 ± 10.33.1 ± 1.8 6.4 ± 2.5 10.9 ± 3.2 (ng/cm²) Average values and standarddeviations of three measurements.

In summary, a new class of triazole-zwitterionic hydrogels that ismechanically robust and FBR-resistant have been designed andsynthesized. Compared to conventional zwitterionic hydrogels which aretypically weak or brittle, these novel TR-ZW hydrogels exhibited highresilience including higher stretchability and bettercompression-resistance or folding-resistance. They retained antifoulingcharacteristics, expected for zwitterionic materials. More importantly,upon subcutaneous implantation in immunocompetent mice, the TR-ZWhydrogels mitigated the fibrosis and promoted the blood vesselformation. The combination of mechanical and FBR-resistant properties ishighly desirable for many biomedical applications such as cellencapsulation and implant modifications, both of which require materialstability and integration with the body for the long-term function andperformance.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1.-36. (canceled)
 37. A monomer of Formula (III):

wherein X¹ is absent or is

Y¹ is

Z¹ is

m₄ is 1 to 50; m₅ is 0 to 10; m₆ is 1 to 50; R″ is H or C₁₋₆ alkyl; R⁴is C₁₋₂₀ alkyl; R⁵ is C₁₋₂₀ alkyl; and R⁶ is C₁₋₂₀ alkyl, with theproviso that when X¹ is absent, Y¹ is not


38. The monomer of claim 37, which has Formula (IIIa):


39. The monomer of claim 37, which has Formula (IIIb):


40. The monomer of claim 37, which has Formula (IIIc):


41. The monomer of claim 37, selected from the group consisting of:


42. A polymeric network comprising crosslinked monomers of Formula(III):

wherein X¹ is absent or is

Y¹ is

Z¹ is

m₄ is 1 to 50; m₅ is 0 to 10; m₆ is 1 to 50; R″ is H or C₁₋₆ alkyl; R⁴is C₁₋₂₀ alkyl; R⁵ is C₁₋₂₀ alkyl; and R⁶ is C₁₋₂₀ alkyl, with theproviso that when X¹ is absent, Y¹ is not


43. The polymeric network of claim 42, wherein the crosslinked monomersof the network are the same.
 44. The polymeric network of claim 42,wherein the crosslinked monomers of the network are different.
 45. Thepolymeric network of claim 42, wherein the monomers are cross-linkedwith a carboxybetaine diacrylamide cross-linker (CBAAX).
 46. A hydrogelcomprising the polymeric network of claim
 42. 47. A capsule comprising:the hydrogel of claim 46 and a therapeutic agent encapsulated in saidhydrogel.
 48. The capsule of claim 47, wherein said therapeutic agentcomprises a preparation of cells.
 49. The capsule of claim 47 furthercomprising: a preparation of cells encapsulated in the hydrogel, whereinthe therapeutic agent is an agent that is released from the preparationof cells.
 50. The capsule of claim 49, wherein the preparation of cellscomprises a preparation of islets.
 51. The capsule of claim 47, whereinthe therapeutic agent is selected from the group consisting of atherapeutic protein, peptide, antibody or binding fragment thereof,antibody mimetic, nucleic acid, small molecule, hormone, growth factor,angiogenic factor, cytokine, anti-inflammatory agents, and anycombination thereof.
 52. A method of delivering a therapeutic agent to asubject, said method comprising: administering the capsule according toclaim 47 to the subject.
 53. The method of claim 52, wherein saidadministering comprising: implanting the capsule into the subject.
 54. Amethod of treating a diabetic subject, said method comprising:implanting the capsule according to claim 50 into a subject withdiabetes.